AbstractSubstance P is a neuropeptide which is present in peripheral C nerve endings and released from them. Free nerve endings of C nerve are present in human epidermis. The effects of substance P on the transmembrane signaling system of pig epidermal sheets were previously reported. In these studies, a small amount of cells other than keratinocytes contaminated the epidermal sheets and the species difference from human was also noticed. Therefore we investigated the effects of substance P on cultured normal human epidermal keratinocytes. Alteration of intracellular free calcium (Ca2+) in single living keratinocytes was studied using an inverted fluorescence microscope and Ca2+‐sensitive dye, Fura 2‐AM. Treatment of normal human epidermal kertinocytes with substance P resulted in an increase in inositol 1,4,5‐trisphosphate and in intracellular Ca2+. Substance P inhibited DNA synthesis of the keratinocytes in a dose‐dependent manner. These results are consistent with the view that substance P stimulates phosphatidylinositol‐4,5‐bisphosphate hydrolysis of human keratinocytes, resulting in inositol 1,4,5‐trisphospha