Although epidermal keratinocytes contain significant amounts of calmodulin, the exact role of calmodulin in epidermal biological activity remains to be determined. Pig-skin (epidermal) calmodulin was purified to homogeneity by DEAE/Sepharose- and phenothiazine-affinity-column chromatography. The characteristics of the purified calmodulin proved to be in good agreement with those of calmodulin obtained from other sources. Phenothiazines (trifluoperazine and chlorpromazine), mepacrine, propranolol, and colchicine inhibited the effect of the purified epidermal calmodulin on the calmodulin-deficient phosphodiesterase of bovine heart. These calmodulin antagonists all had inhibitory effects on the thymidine incorporation of pig-skin epidermal keratinocytes. These observations support the assumption that calmodulin might play an important role in epidermal keratinocyte proliferation.