Reversal of specific cell-cell adhesions between allogeneic cytolytic T lymphocytes (CTL) and 51Cr-labeled target cells has been studied by using a functional assay for specific target cell binding. Functional adhesions between CTL and a tumor cell target, the P815 mastocytoma, were stable in suspension for greater than 5 hr. Addition of unlabeled target cells, however, resulted in rapid reversal of functional binding. This reversal appeared to be specific in that no reversal was induced by tumor cells of other H-2 types. Functional reversal of conjugates between CTL and labeled spleen cells syngeneic with P815 also occurred in the presence of unlabeled P815 cells or normal spleen cells of the appropriate H-2 type. The reversal by P815 was substantially more efficient than by normal spleen cells. Furthermore, normal spleen cells of the appropriate H-2 type did not induce reversal of CTL-P815 target conjugates. Purified plasma membranes from P815 cells did not specifically reverse functional binding of CTL to either P815 or normal spleen cell targets. These results demonstrate that functional CTL-target cell binding can be specifically reversed by interaction with free target cells. The rate of reversal appears to be dependent on the relative affinity of the CTL for the bound versus the free target cell. CTL may provide a useful system for studying reversal of specific cell-cell adhesions.