An alloactivated secondary mixed lymphocyte reaction (MLR) culture (SMC), which was suppressive in the MLR with autologous responder cells, was studied in more detail. In particular, we investigated whether the suppressive activity of this SMC was mediated by cytotoxic T cells or whether bona fide suppressor cells were involved. The SMC suppressed an MLR when the stimulator cells shared Bw57, Bw60, or Dw19 with the original stimulator. Separation of the SMC into CD4+ and CD8+ fractions demonstrated that the CD8+ fraction contained suppressive and cytotoxic activity against Bw57 or Bw60 antigens, while the CD4+ fraction contained both activities against the Dw19 specificity. The CD8+ fraction was also suppressive in the reverse MLR, while the CD4+ fraction was not. Limiting dilution analysis demonstrated that all CD8+-suppressive cultures were also cytotoxic, whereas in the CD4+ fraction both cytotoxic and non-cytotoxic suppressor cultures were found.