To investigate the specificity of anti HI antibodies peptides from the N- and C-domain of HI and the synthetic oligonucleotide (AT)6were complexed. Circular dichroism (CD) spectroscopy indicated that the free peptides H1(1-16), H1(204-218) and C(121-210) in low salt buffer assume a random structure but become helical when bound to the oligonucleotide. The structured and unstructured HI fragments were then analyzed by enzyme linked immunosorbent assay (ELISA) with anti-Hl antibodies in sera from patients with systemic lupus erythematosis (SLE) and with the monoclonal anti-Hl antibody MRA-12 derived from MLR lpr/lpr autoimmune mice. Binding of these antibodies to HI(204-218) and C was inhibited to a level of 50% when these HI peptides were complexed with (AT)6. When the same antibody was tested with HI fragment GC(34-210), attachment to oligonucleotide (AT)6did not influence antibody binding. Competition studies with liquid phase GC and C antigen against solid phase GC and C indicated that liquid phase GC was more efficient in displacing antibody binding reactivity than liquid phase C. The displacement effect of both liquid phase antigens was greatest against solid phase C. We conclude that anti-Hl autoantibodies are directed against an epitope located near the junction of the G- and C-domain which is exposed and not masked when HI is bound to DNA.