Induction of growth arrest and monocyte differentiation of HL-60 leukemia cells by 1α,25 dihydroxyvitamin D 3 (1α,25(OH) 2D 3) is well established. By contrast, we have observed, that 1α,25(OH) 2D 3 and its metabolites play separate roles in clonal expansion and survival of differentiating HL-60 cells. Cells that had differentiated by 48 h (CD14 positive) grew slower than control cells, whereas CD14 negative cells were growing faster at this time point. Inhibiting 1α,25(OH) 2D 3 or 1α,25(OH) 2-20-epi-D 3 metabolism, by the 25(OH)D 3-24-hydroxylase inhibitor ketoconazole, abolished hyperproliferation of CD14 negative cells. Instead, both the onset of differentiation and subsequent apoptosis were enhanced. These events were associated with immediate up-regulation of the cyclin-dependent kinase inhibitor p21 ( waf1) and a lack of sustained expression, respectively. Stimulation and inhibition of growth by vitamin D 3-related compounds was observed to be concentration and metabolite specific. Low amounts of 1α,25(OH) 2-20-epi-D 3 and 1α,24,25(OH) 3-20-epi-D 3 stimulated HL-60 cell growth. At higher concentrations, 1α,25(OH) 2-20-epi-D 3 was a more potent inducer than 1α,24,25(OH) 3-20-epi-D 3 of HL-60 differentiation; 1α,25(OH) 2-20-epi-24-oxo-D 3 was exclusively pro-differentiative at all concentrations. 1α,25(OH) 2-20-epi-D 3 and 1α,24,25(OH) 3-20-epi-D 3 stimulated proliferation of KG-1a leukemia cells, but neither of these compounds nor 1α,25(OH) 3-20-epi-24-oxo-D 3 exerted pro-differentiative effects on these cells. These findings shed new light on the pro- and anti-proliferative effects of 1α,25(OH) 2D 3 and lead to the postulate that metabolism of 1α,25(OH) 2D 3 and its 20-epi analog regulates different subsets of genes so as to co-ordinate population expansion and the differentiation process. Furthermore, 1α,25(OH) 2D 3 metabolism and/or sensitivity to the effects of metabolites may be altered in transformed cells to derive a clonal advantage.