Although calmodulin has been suggested as an important regulator of keratinocyte proliferation, its precise role remains unknown. We employed a calmodulin antagonist, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), to examine the role of calmodulin on keratinocyte proliferation. N-(6 aminohexyl-1-naphthalenesulfonamide (W-5), a chlorine-deficient analogue of W-7 with little anti-calmodulin activity, was used as the control. W-7 markedly inhibited thymidine incorporation of pig epidermis at concentrations close to its anti-calmodulin activity; W-5 had no effect on the thymidine incorporation. The inhibitory effect of W-7 was reversible; the removal of W-7 from the incubation medium resulted in the reinitiation of the thymidine incorporation, suggesting that W-7 is not a cytotoxic agent. These results are consistent with the view that calmodulin is an essential regulator of keratinocyte proliferation. The epidermal beta-adrenergic response, which is decreased in various hyperproliferative epidermal abnormalities, was increased in W-7-treated hypoproliferative epidermis. The epidermal SOD activity, which is also decreased in the hyperproliferative epidermis, however, was not affected by the W-7 treatment.