We have previously shown that keratinocytes in vitro can convert biologically inactive vitamin D₃ to the hormone calcitriol (1α,25-dihydroxyvitamin D₃). This study was initiated to test whether the ultraviolet-B-induced photolysis of provitamin D₃ (7-dehydrocholesterol), which results in the formation of vitamin D₃, can generate calcitriol in an in vivo-like human skin equivalent model made of fibroblasts in a collagen matrix as the dermal component and keratinocytes as the epidermal component. Cultures were preincubated with increasing concentrations of 7-dehydrocholesterol (0.53–5.94 nmol per cm² human skin equivalent) at 37°C and irradiated with monochromatic ultraviolet B at wavelengths ranging from 285 to 315 nm (effective ultraviolet doses 7.5–45 mJ per cm²). In our in vitro model irradiation with ultraviolet B resulted in a sequential metabolic process with generation of previtamin D₃ followed by the time-dependent formation of vitamin D₃, 25-hydroxyvitamin D₃, and ultimately calcitriol in the femtomolar range. Unirradiated cultures and irradiated cultures without keratinocytes generated no calcitriol. Irradiation of skin equivalents at wavelengths > 315 nm generated no or only trace amounts of calcitriol. The ultraviolet-B-triggered conversion of 7-dehydrocholesterol to calcitriol was strongly inhibited by ketoconazole indicating the involvement of P450 mixed function oxidases. The amount of calcitriol generated was dependent on the 7-dehydrocholesterol concentration, on wavelength, and on ultraviolet B dose. Hence, keratinocytes in the presence of physiologic concentrations of 7-dehydrocholesterol and irradiated with therapeutic doses of ultraviolet B may be a potential source of biologically active calcitriol within the epidermis.