A competitive inhibition assay was used to define the specificity of the in vitro cytotoxicity of activated lymphocytes. Human and mouse effector cells were produced by prestimulation with phytohemagglutinin, pokeweed mitogen, or purified protein derivative for several days. The addition of increasing numbers of unlabeled Chang cells to a fixed number of stimulated human lymphocytes and 51Cr-labeled Chang cells gradually decreased chromium release. Admixture of unlabeled human bladder tumor cells, lung cells, or monkey kidney cells had a similar effect, whereas mouse L cells were not inhibitory. The reverse was found to be true when stimulated mouse lymphocytes and 51Cr-labeled mouse L cells were incubated. In this case, the addition of unlabeled L or rat tumor cells effectively inhibited 51Cr release, whereas human or monkey cells were less inhibitory. Both human and mouse cells inhibited the cytotoxicity of human effector lymphocytes towards mouse L cells. The cytotoxic effects of mouse lymphocytes on human cells were also decreased by addition of unlabeled human, monkey, or mouse cells, but in this case human cells were most inhibitory. The results indicate that activated lymphocytes recognize several surface structures on the target cells. Some of these structures may be shared by cells of different species origin, whereas others seem to be unique for cells from phylogenetically closely related species.