We have characterized the antiproliferative effects of the phenothiazines, a group of antipsychotic drugs possessing a wide range of pharmacological actions. The phenothiazines inhibited both the proliferation and clonogenicity of L1210 leukemic lymphocytes. This effect was dependent on both time of exposure and concentration of drug. Clonogenicity of cells in the logarithmic phase of growth was inhibited by greater than 99% at a concentration of drug that had no effect on cells in the plateau phase of growth. Human and murine cell lines, grown either in suspension or in monolayers, were equally susceptible. Calmodulin (CaM), purified from L1210 cells by preparative polyacrylamide gel electrophoresis, had sensitivity to inhibition by phenothiazines similar to that reported for CaM prepared from brain. The order of potency was trifluoperazine greater than or equal to fluphenazine greater than chlorpromazine greater than chlorpromazine-sulfoxide. As a class, these drugs were less potent antagonists of CaM than was the bee venom polypeptide, melittin. The antiproliferative effects of phenothiazines were similar to the anticalmodulin effects. Thus, the same order of potencies was seen for both effects; the shapes of the dose-response curves were similarly steep and the effects of excess calcium on the inhibition of both were identical. These studies add pharmacological support for CaM being a potential intracellular target for the antiproliferative effect of the phenothiazines.