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 1alpha,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-D3-24-hydroxylase (CYP24A1), active vitamin-D3-25-hydroxylase (CYP27A1), and 25-hydroxyvitamin-D3-1alpha-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.