Vitamin D3 can be hydroxylated sequentially by cytochrome P450scc (CYP11A1) producing 20-hydroxyvitamin D3, 20,23-dihydroxyvitamin D3 and 17,20,23-trihydroxyvitamin D3. The aim of this study was to characterize the ability of vitamin D3 to associate with phospholipid vesicles and to determine the kinetics of metabolism of vitamin D3 by P450scc in vesicles and in 2-hydroxypropyl-beta-cyclodextrin (cyclodextrin). Gel filtration of phospholipid vesicles showed that the vitamin D3 remained quantitatively associated with the phospholipid membrane. Vitamin D3 exchanged between vesicles at a rate 3.8-fold higher than for cholesterol exchange and was stimulated by N-62 StAR protein. The Km of P450scc for vitamin D3 in vesicles was 3.3 mol vitamin D3/mol phospholipid and the rate of conversion of vitamin D3 to 20-hydroxyvitamin D3 was first order with respect to the vitamin D3 concentration for the range of concentrations of vitamin D3 that could be incorporated into the vesicle membrane. 20-Hydroxyvitamin D3 was further hydroxylated by P450scc in vesicles, producing primarily 20,23-dihydroxyvitamin D3, with Km and kcat values 22- and 6-fold lower than those for vitamin D3, respectively. 20,23-dihydroxyvitamin D3 was converted to 17,20,23-trihydroxyvitamin D3 with even lower Km and kcat values. Vitamin D3 and cholesterol were metabolized with comparable efficiencies in cyclodextrin, but the Km for both showed a strong dependence on the cyclodextrin concentration, decreasing with decreasing cyclodextrin. This study shows that vitamin D3 quantitatively associates with phospholipid vesicles, can exchange between membranes, and can be hydroxylated by membrane-associated P450scc but with lower efficiency than for cholesterol hydroxylation. The kcat values for metabolism of vitamin D3 in vesicles and 0.45% cyclodextrin are similar, but the ability to solubilize vitamin D3 at a concentration higher than its Km makes the cyclodextrin system more efficient for producing the hydroxyvitamin D3 metabolites for further characterization.