Vitamin D-3 plays an important role in the control of cell proliferation and differentiation. Cholesterol 25-hydroxylase (CH25H) is an enzyme converting cholesterol into 25-hydroxycholesterol. Vitamin D3 as well as 25-hydroxycholesterol has been shown to inhibit cell growth and induce cell apoptosis. Here we show that 10 nM 1 alpha,25(OH)(2)D-3 and 500 nM 250HD(3) upregulate CH25H mRNA expression in human primary prostate stromal cells (P29SN). Protein synthesis inhibitor cycloheximide does not block 1 alpha,25(OH)(2)D-3 mediated upregulation of CH25H mRNA. Transcription inhibitor actinomycin D blocks basal level as well as lot,25(OH)(2)D-3 induced CH25H mRNA expression. lot,25(OH)(2)D-3 has no effect on CH25H mRNA stability. 25-Hydroxycholesterol significantly decreased the P29SN cell number. A CH25H enzyme inhibitor, desmosterol, increases basal cell number but has no significant effect on vitamin D-3 treated cells. Our data suggest that ch25h could be a vitamin D-3 target gene and may partly mediate anti-proliferative action of vitamin D-3 in human primary prostate stromal cells. (c) 2006 Elsevier Inc. All rights reserved.