The nature of the Ca2+ requirement of lymphocyte-mediated cytolysis (LMC) has been explored pharmacologically with a number of putative calmodulin antagonists. N-(6-Aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), N-(6-aminohexyl)-1-naphthalenesulfonamide (W-5), trifluoperazine, and chlorpromazine were found to inhibit LMC (IC50 values = 8.9, approximately 50, 7.4, and 9.4 microM, respectively) at concentrations which were not detectably toxic to either the effector or the target cell. Pimozide inhibited LMC by 50% at 15 microM but caused a substantial decrease in lymphocyte ATP content and viability at this concentration. 1-[Bis(p-chlorophenyl)methyl]-3-[2,4-dichloro-beta-(2,4-dichlorobenzy loxy) phenethyl]imidazolium chloride (R 24 571, calmidazolium), which has been reported to be the most potent antagonist of isolated calmodulin, caused a marked decrease in lymphocyte ATP content and viability at concentrations greater than 4 microM and inhibited LMC only slightly at similar concentrations. Trifluoperazine sulfoxide and chlorpromazine sulfoxide were not inhibitory to LMC at less than or equal to 20 microM. LMC was inhibited in a sustained manner when cytolytic lymphocytes, but not target cells, were pretreated separately with W-7 or chlorpromazine at 37 degrees and were then washed free of exogenous drug prior to the start of the LMC assay. The above cellular effects of the calmodulin antagonists were reduced in magnitude when the serum concentration in the culture medium was increased (from 5% to 20%). The inhibition of LMC by micromolar concentrations of W-7, trifluoperazine, and chlorpromazine, as well as the relative inactivities of W-5 versus W-7 and of the sulfoxide derivatives of trifluoperazine and chlorpromazine, are consistent with calmodulin's being a lymphocyte receptor whose occupancy by Ca2+ is required for the performance of this cytolytic function. However, this conclusion must be tempered by the finding that even W-7, trifluoperazine, and chlorpromazine can exert nonspecific effects on the energy metabolism and viability of the cytolytic lymphocytes at concentrations of drug severalfold higher than those required to inhibit LMC.