Changes in the concentration of extracellular calcium affect the balance between proliferation and differentiation in epidermal keratinocytes. Undifferentiated keratinocytes respond to the acute increase in the concentration of extracellular calcium with an increase of intracellular calcium concentration and inositol trisphosphate production, and, subsequently, the expression of differentiation related genes. Our previous studies demonstrated the presence of a calcium-sensing receptor in human keratinocytes, which is identical to the parathyroid calcium-sensing receptor. In this study we showed that the calcimimetic compound NPS R-467, a selective calcium-sensing receptor activator, augmented the calcium-elicited inositol trisphosphate response of cloned human keratinocyte calcium-sensing receptor expressed in human embryonic kidney cells 293. In order to define the role of the calcium-sensing receptor in calcium induced epidermal differentiation, we investigated the ability of NPS R-467 to raise intracellular Ca2+ and stimulate differentiation in normal human foreskin keratinocytes. In the presence of 0.03 mM Ca2+, NPS R-467 increased the intracellular calcium concentration response in a concentration-dependent fashion. Undifferentiated normal human foreskin keratinocyte cells responded to increased extracellular calcium concentration with increased intracellular calcium concentration. NPS R-467 potentiated this response by increasing the maximal response. Its stereoisomer, NPS S-467, was not active in raising intracellular calcium concentration. Increasing extracellular calcium concentration from 0.03 to 1.2 mM stimulated the promoter activity of the differentiation marker gene, involucrin. NPS R-467 potentiated the calcium-stimulated increase in involucrin promoter activity unlike NPS S-467 or vehicle. Northern analysis of the normal human foreskin keratinocyte cells treated with NPS R-467 demonstrated potentiation of the calcium-stimulated increases in involucrin and transglutaminase mRNA levels. These results support the hypothesis that the calcium-sensing receptor expressed in keratinocytes mediates at least part of the intracellular calcium response to extracellular calcium and calcium-induced differentiation.