Vitamin D and its analogues are potent regulators of cell growth and differentiation both in vivo and in vitro. We studied the effects of 25-hydroxyvitamin D3 [25(OH)D3], 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] and vitamin D analogue, EB 1089, on the growth of a human ovarian cancer cell line, OVCAR-3. We also studied the expression of vitamin D metabolising enzymes 24-hydroxylase (24OHase) and 1α-hydroxylase (1αOHase). Our results showed that high concentrations (10 and 100 nM) of 1,25(OH)2D3 inhibited a cell proliferation, whereas low concentration (0.1 nM) stimulated growth of the OVCAR-3 cells. In the concentration range of 10–500 nM a prohormone, 25(OH)D3, stimulated growth. An amount of 1 nM EB 1089 and 100 nM 1,25(OH)2D3 inhibited growth with an equal magnitude. The expression of 24OHase was strongly induced by 1,25(OH)2D3 and EB 1089 in OVCAR-3 cells, and analysis of vitamin D metabolites showed the functionality of 24OHase. An inhibition of 24OHase activity with a novel 24OHase inhibitor enhanced growth-inhibiting effects of 1,25(OH)2D3 and suppressed the growth stimulation of 100 nM 25(OH)D3. We also report the expression of a vitamin D activating enzyme, 1αOHase, in 7 ovarian cancer cell lines. The production of 1,25(OH)2D3 in OVCAR-3 cells was low, possibly due to an extensive activity of 24OHase or a low 1αOHase activity. These results suggest that in ovarian cancer cells vitamin D metabolizing enzymes might play a key role in modulating the growth response to vitamin D. The possible mitogenic effects of vitamin D should be considered when evaluating treatment of ovarian cancer with vitamin D. © 2003 Wiley-Liss, Inc.