Abstract The effects of papain, trypsin, Pronase, neuraminidase, and phospholipase C on in vitro cell-mediated cytotoxicity (CMC) and antibody-dependent cell cytotoxicity (ADCC) have been studied in a syngeneic tumor system. Proteolytic enzyme treatment of lymphoid (effector) cells at low concentrations (<0.1 mg/ml) and for short times (less than 30 min) produced a 2- to 6-fold increase in CMC and ADCC. Neuraminidase also augmented both CMC and ADCC, whereas phospholipase C had no effect. The specificity of the cytotoxicity (CMC) amplification following enzymatic treatment was indicated by observations that: a) the activated immune cells were not cytotoxic to unrelated tumor cells, and b) normal cells were not activated. The enzymatically induced potentiation was observed only with organs which already had demonstrable cytotoxic activity. Kinetic studies indicated that the enzymatic treatment may produce a quantitative as well as a qualitative change in the lymphoid cell population. Enzymatic treatment of lymphoid cells may be a valuable means of increasing the sensitivity of in vitro assays for cell-mediated immunity. Pretreatment of lymphoid cells with high enzyme concentrations (over 1 mg/ml) or for prolonged periods of time (over 1 hr) did not reduce ADCC, but strongly suppressed CMC. This difference in response to proteolytic enzymes emphasizes chemical differences in the membrane components responsible for CMC vs ADCC.