To formally test the hypothesis that allospecificity of the human mixed lymphocyte response-generated suppressor cells is restricted to the major histocompatibility complex, 18 primary mixed lymphocyte responses, using peripheral blood lymphocytes of 10 HLA-typed families, were used to generate suppressor effector cells. Responder lymphocytes shared one HLA haplotype with the stimulator cells. At day 10, the effector cells were tested for their ability to suppress proliferation of 55 test mixed lymphocyte responses consisting of naive autologous lymphocytes as responders and irradiated cells from individual family members as stimulators. Suppression was predicted when the test culture stimulators expressed the same HLA haplotype to which the suppressor cells were primed. There was statistically significant correlation between suppression and HLA haplotype sharing (p = 0.0143). In 6 out of 13 assays, however, we observed suppression that was not predicted on the basis of relevant HLA haplotype sharing. These stimulators shared only HLA-A2 with the priming haplotype. HLA antigen sharing was not seen in the 7 remaining, nonsuppressed cultures. We conclude that, within families, the effector function of in vitro generated suppressor cells is restricted by the major histocompatibility complex.