In previous work, lysis of allotargets was routinely observed with PBL from nonimmune channel catfish. In the work reported here, greatly increased (approximately 100-fold) cytotoxic responses were generated by stimulation of channel catfish PBL with irradiated cells of allogeneic cloned B cell lines in mixed leukocyte cultures (MLC). This increased cytotoxicity did not appear to be simply a consequence of cell proliferation since stimulation of catfish PBL proliferative responses with polyclonal mitogens did not result in increased lysis. Somewhat surprisingly, the MLC-generated cytotoxicity did not exhibit allospecificity; i.e., allogeneic targets from other fish were as susceptible to lysis as were the cells used as stimulators. This apparent lack of allospecificity in MLC-generated cytotoxicity was confirmed by "cold" target inhibition assays. However, autologous targets were not killed, clearly demonstrating that MLC-generated effectors could distinguish "self" from "nonself" at the level of lysis/recognition. Although their origin is unresolved, the MLC-generated effectors may be a source of highly enriched fish cytotoxic cells and thus facilitate directly addressing questions pertaining to the evolution of such cells.