The tryptophan dimer 1,1′-ethylidenebis[L-tryptophan] was identified as a contaminant of tryptophan preparations associated with Eosinophilia-Myalgia Syndrome. In this paper, we describe experiments examining the hypothesis that 1,1′-ethyli-denebis[L-tryptophan] acts as an amino acid analog replacing L-tryptophan during the synthesis of proteins. We propose further that proteins containing 1,1′-ethylidenebis[L-tryptophan] are rejected in an autoimmune process identified clinically as Eosinophilia-Myalgia Syndrome.Rabbit reticulocyte lysates containing an estimated 1μM L-tryptophan were used to assay the ability of 1,1′-ethylidenebis[L-tryptophan] to compete with3H-L-tryptophan for incorporation into proteins translated from BMV RNA. 1,1′-Ethylidenebis[L-tryptophan] in concentrations of 40, 80 and 110μM reduced lysate3H-L-tryptophan incorporation to 81%, 76% and 75% of control incorporation obtained in the absence of 1,1′-ethylidenebis[L-tryptophan]. In the presence of 20μM L-tryptophan, 110μM 1,1′-ethylidenebis(L-tryptophan] reduced3H-L-tryptophan incorporation to 56% of control incorporation. In contrast, ethyl-L-tryptophan did not significantly reduce3H-L-tryptophan incorporation. In the presence of 110μM 1,1′-ethylidenebis[L-tryptophan] and 20μM L-tryptophan,3H-L-leucine incorporation was not significantly reduced compared to incorporation in the absence of 1,1′-ethylidenebis[L-tryptophan], demonstrating that proteins were translated to full length during elongation. These findings suggest that 1,1′-ethylidenebis[L-tryptophan], but not ethyl-L-tryptophan, reduced3H-L-tryptophan incorporation into proteins by substituting for L-tryptophan rather than by causing premature termination or significant slowing of nascent protein chains.In further experiments, the ethylidene bridge of 1,1′-ethylidenebis[L-tryptophan] was labeled during synthesis with14C-acetaldehyde.14C-1,1′-ethylidenebis[L-tryptophan] was added to rabbit reticulocyte lysates containing BMV RNA to a concentration of 110μM 1,1′-ethylidenebis[L-tryptophan]. On SDS-PAGE gels, bands of14C-labeled proteins were detected at the 5 band positions expected from BMV RNA; i.e., at approximately 100, 97, 35, 20 and 15 kD. We conclude from these experiments that trytophanyl t-RNA synthetase can activate and attach 1,1′-ethylidenebis[L-tryptophan] to tryptophanyl-t-RNA and that 1,1′-ethylidenebisll-tryptophan] can be incorporated into proteins in place of L-tryptophan.To determine whether Eosinophilia-Myalgia Syndrome was associated with sensitization to 1,1′-ethylidenebis[L-tryptophan], we constructed artificial haptens to mimic proteins containing 1,1′-ethylidenebis[L-tryptophan] residues. Haptens were synthesized by coupling 1,1′-ethylidenebis[L-tryptophan], ethyl-L-tryptophan or L-tryptophan to albumin with glutaraldehyde. Peripheral blood lymphocytes were isolated from patients stably recovered from Eosinophilia-Myalgia Syndrome for 2 yr or more and used in proliferation assays against lymphocytes from controls to determine if they were immunosensitive to synthesized haptens. Only one out of the five recovered Eosinophilia-Myalgia Syndrome patients demonstrated significant reactivity to the synthesized hapten containing 1,1′-ethylidenebis[L-tryptophan]. This patient also demonstrated an increased reactivity to the synthesized hapten containing L-tryptophan. A second recovered Eosinophilia-Myalgia Syndrome patient demonstrated increased sensitivity to the synthesized hapten containing ethyl-L-tryptophan. These results provide inconclusive evidence that incorporated 1,1′-ethylidenebis[L-tryptophan] provides an immunogenic stimulus. However, the synthesized haptens may not have sufficiently mimicked the configurations of proteins containing 1,1′-ethylidenebis[L-tryptophan] residues to test for sensitization or sensitization could have been lost in recovered patients following corticosteroid treatment and the turnover of affected proteins.