Supernatant derived from the incubation of normal, unstimulated spleen cells was able to stimulate a strong specific in vitro cytotoxic lymphocyte (CTL) response in allogeneic spleen cells primed with the corresponding haplotype. The supernatant antigen (SA) was as efficient in inducing secondary CTLs as equivalent numbers of irradiated, adherent cell-depleted spleen cell stimulators present during the culture period, and the CTL stimulation with both was dependent on the presence of responder splenic adherent cells. SA obtained from P815 tumour cells was unable to stimulate a similar response under the same conditions. The stimulating fraction of the SA showed the characteristics of lipid-associated major histocompatibility (MHC) antigens shed from viable lymphocytes, in that it was removed with specific H-2 antiserum, it was sedimented at 100,000 g and its activity was reduced if spleen cells were incubated at 4 degrees instead of 37 degrees. These results indicate a possible role for the shedding of MHC antigens in the general induction of a cytotoxic response.