Alloantisera that enhance survival of heart allografts in inbred rats were tested for their effect on mixed leukocyte reactions (MLR). Lewis (L) rats, administered 107Brown Norway (BN) rat bone marrow or spleen cells i.v. 7 days prior to transplantation, exhibited significant prolongation of BN graft survival. Passive administration to normal L rats of serum obtained from L rats 7 days after such i.v. immunization similarly prolonged BN graft survival. The unidirectional MLR of normal or presensitized L spleen cells with irradiated BN spleen cells was also suppressed by this serum in comparison with MLRs containing normal L serum. Twenty percent alloantiserum decreased the MLR of unsensitized L with irradiated BN as much as 6-fold. Less suppression was demonstrated with 10% antiserum and none with 5%. Alloantiserum suppression of MLR remained demonstrable in the absence of hemolytic complement. Immuno-logical specificity was demonstrated because “enhancing” serum suppressed the MLR of L to irradiated ACI rat leukocytes to a much smaller degree. When splenic leukocytes from presensitized L rats were washed free of enhancing serum and then incubated in normal L serum, they showed increased proliferative activity in MLR with irradiated BN spleen cells. Since these L cells, after removal of enhancing serum, were very active in the MLR, it indicates that enhancement by serum factors rather than central tolerance was the mechanism of graft prolongation. Thus, inhibition of MLR might be useful as an assay for the presence of enhancing alloantiserum.