Human keratinocytes are fully competent cells of the vitamin D (VD) hormone system. They have the capacity to generate VD, to convert it to hormonally active 1α,25(OH) 2D 3 and subsequently, to metabolize the hormone by self-induced CYP24. These reactions generate a cascade of highly transient products and, eventually terminate biologic activity. To elucidate regulatory principles in the VD cascade in more detail, we made use of novel selective CYP24 inhibitors, recently synthesized by our group. Here, we describe the effects of VID400 and SDZ 89–443 on the metabolism of 20 nM 3H-25(OH)D 3 in human keratinocytes, analyzed by sensitive HPLC methods. First, we present evidence that freshly generated 1α,25(OH) 2D 3 does not down-regulate 1α-hydroxylation, as commonly assumed. The transient time course of 1α,25(OH) 2D 3, could be explained by its fast 24-hydroxylation to polar products, undetectable by usual HPLC-analysis of organic extracts. On inhibition of CYP24, 1α-hydroxylation continued throughout extended periods, indicating its constitutive nature. Asking whether 1α,25(OH) 2D 3 derived metabolites [1α,25(OH) 2-3epi-D 3, 1α,24(R),25(OH) 3D 3, 1α,25(OH) 2-24oxo-D 3, 1α,23(S),25(OH) 3-24-oxo-D 3 and calcitroic acid] would regulate 1α-hydroxylase, we pre-treated cells with 20 nM of these metabolites for 5 h and 24 h. Subsequent incubation with 3H-25(OH)D 3 demonstrated that neither metabolite substantially impaired 1α-hydroxylase, while all of them transiently induced CYP24 activity. Analyzing the effects of VID400 on the kinetics of 3H-25(OH)D 3, we showed that 1α-hydroxylation rather than 24-hydroxylation was rate-limiting in the C-24 oxidation pathway - again suggesting constitutive expression of 1α-hydroxylase. CYP24 inhibitors effectively increased the levels and lifetime of all transient 1α-hydroxylated metabolites, especially of 1α,25(OH) 2-3epi-D 3 that became the predominant lipid soluble metabolite. Highly increased levels of 1α,23(S),25(OH) 3-24-oxo-D 3, the metabolite preceding side chain cleavage, indicated involvement of CYP24 also in the terminal step of the cascade. Besides using inhibitors of CYP24 as tools to explore mechanisms in the VD cascade, they also appear to be valuable to discover the intrinsic biologic functions of distinct metabolites.