Earlier investigations in our laboratory have demonstrated that UVB irradiation of cultured human keratinocytes induces the conversion of 7-dehydrocholesterol (7-DHC) to hormonally active 1α,25-dihydroxyvitamin D₃ (calcitriol). In the research presented here, we have investigated the influence of UVB-triggered calcitriol production on gene expression of the vitamin D₃ hydroxylating enzymes catabolic 25-hydroxyvitamin-D ₃-24-hydroxylase (CYP24A1), active vitamin-D₃-25- hydroxylase (CYP27A1), and 25-hydroxyvitamin-D₃-1α-hydroxylase (CYP27B1) using real-time PCR. Our results demonstrate a marked and wavelength-dependent induction of CYP24A1-mRNA in cultured human keratinocytes supplemented with 7-DHC, which parallels the spectral optimum at about 300 nm of calcitriol production as detected by HPLC and radioimmunoassay. Owing to the high sensitivity of real-time PCR, we provide evidence of a wavelength-dependent induction of CYP24A1-mRNA even in 7-DHC-deficient keratinocytes. Interestingly, we have found a strong but transient induction of CYP24A1-mRNA in non-irradiated keratinocytes, followed by accelerated cell proliferation. In contrast, UVB and calcitriol had no effect on gene expression of CYP27A1 and CYP27B1. We conclude from these experiments a constitutive gene expression of the vitamin D₃ hydroxylases, whereas the catabolic enzyme CYP24A1 is markedly regulated by UVB, calcitriol, and perhaps cell proliferation. If confirmed at protein level, these findings could have an impact on epidermal vitamin D₃ metabolism and its modulation by UVB in health and disease.