Rat antisera prepared against murine, periodate-activated alloimmune cytotoxic lymphocytes (termed RAT) have previously been shown to effectively block T-cell-mediated cytotoxicity (CMC) at the "lethal hit" stage of cytolysis (J.C. Hiserodt and B. Bonavida, J. Immunol. 126, 256, 1981). Both natural killer (NK) and cytotoxic T lymphocytes (CTL) have been shown to mediate lysis by the same pathway, namely binding of effector to target cells, programming for lysis, and killer cell-independent target cell lysis. This result suggested that the molecular mechanism of NKCMC and CTLCMC may also be similar. In this context, RAT-mediated blocking of CTL was examined for its ability to block NKCMC. The results show that (1) addition of RAT serum or IgG fractions blocked NKCMC in the absence of complement in a 4-hr 51CR-release assay, and blocking was directed at the effector cell; (2) at the single-cell level, RAT serum blocked the formation of conjugates between effector and target cells; (3) in a Ca2+-pulse experiment, in which the effectors and targets were first allowed to bind in the absence of Ca2+ for 1 hr at 37 degrees C, followed by the addition of Ca2+ to initiate the lytic event, RAT was capable of blocking cytotoxicity after conjugate formation at the Ca2+-dependent lethal hit stage of cytolysis. The similarity of results in RAT blocking experiments of both the CTL and NK systems suggests a common molecular mechanism of cytolysis.