Non-toxic concentrations of fluphenazine caused a marked (90%) inhibition of NK-mediated cytolysis of YAC-1 tumor cells. The biologically inactive sulphoxide derivative was not inhibitory and the efficacy of inhibition of other compounds was directly correlated (r = -0.96, p less than 0.02) with their reported affinities for calmodulin. Fluphenazine may act on the earliest stages of the target-effector interaction since conjugate formation between CBA effectors and YAC target cells decreased from 20% to 6% (p less than 0.02) upon pre-treatment with fluphenazine. However, fluphenazine was not selective for NK cells since cytotoxic T lymphocytes, derived from both mixed lymphocyte culture and by concanavalin A stimulation, revealed depressed cytolytic activity against P815 tumor targets after fluphenazine treatment. Tumoricidal activity by activated macrophages and effectors of antibody-dependent cell-mediated cytotoxicity was also blocked. Fluphenazine inhibition was reversible, since addition of 1.25-5 micrograms/ml of the calcium ionophore A23187 to fluphenazine-treated effectors restored NK binding and cytolytic functions to normal levels. Calmodulin was isolated from NK-enriched populations by affinity chromatography on sepharose-fluphenazine columns. Pre-treatment of effector cells with [3H]fluphenazine and isolation of calmodulin by immunoprecipitation and SDS-polyacrylamide gel electrophoresis showed that fluphenazine entered the cells and bound a calmodulin-like molecule. These data are compatible with the suggestion that fluphenazine inhibits NK function by inactivating the calcium-calmodulin complex and thereby altering binding events in the target-effector interaction. Other actions of the phenothiazines are also possible.