The hormonal forms of vitamin D3 (1,25-dihydroxyvitamin D3 and synthetic vitamin D3 analogues) are potent regulators of keratinocyte growth and stimulators of keratinocyte differentiation. Recent experiments in vitro on cultured keratinocytes indicate that Ca2+ may be a second messenger mediating the effects related to the induction of keratinocyte differentiation by the hormonal forms of vitamin D3. In this study we employed the technique of ion capture cytochemistry to investigate the effects of a potent vitamin D3 analogue, KH 1060 (20-epi-22-oxa-24 alpha-homo-26,27-dimethyl-1,25-dihydroxyvitamin D3), on the distribution of bound intracellular and extracellular calcium in murine epidermis in vitro. Topical treatment of the skin with KH 1060 (0.4 mmol/l) resulted in the development of epidermal hyperplasia and hyperkeratosis. We observed that epidermis treated with KH 1060 contained fewer calcium deposits in the upper epidermal strata (both intra- and extracellularly) than the control skin. This phenomenon was rapid and occurred after only a single application of KH 1060. Calcium distribution in the basal cell layer was not affected. We propose that observed reduction in the quantity of calcium deposits was caused by the release of sequestered calcium from the intracellular stores and cellular Ca2+ uptake, leading eventually to the increase in the intra-cellular concentration of ionized calcium. The ability of the active vitamin D3 compounds to release Ca2+ may be important for their activity in psoriasis.