20-Hydroxyvitamin D3 (20(OH)D3), the major product of CYP11A1 action on vitamin D3, is biologically active and is produced in vivo. As well as potentially having important physiological actions, it is of interest as a therapeutic agent due to its lack of calcemic activity. In the current study we have examined the ability of CYP24A1, the enzyme that inactivates 1,25-dihydroxyvitamin D3 (1,25(OH)2D 3), to metabolize 20(OH)D3. Rat CYP24A1 was expressed in Escherichia coli, purified by Ni-affinity chromatography and assayed with substrates incorporated into phospholipid vesicles which served as a model of the inner mitochondrial membrane. In this system CYP24A1 metabolized 1,25(OH)2D3 with a catalytic efficiency 1.4-fold higher than that seen for 25-hydroxyvitamin D3 (25(OH)D3). CYP24A1 hydroxylated 20(OH)D3 to several dihydroxy-derivatives with the major two identified by NMR as 20,24-dihydroxyvitamin D3 (20,24(OH)2D3) and 20,25-dihydroxyvitamin D3 (20,25(OH)2D3). The catalytic efficiency of CYP24A1 for 20(OH)D3 metabolism was more than 10-fold lower than for either 25(OH)D3 or 1,25(OH)2D3 and no secondary metabolites were produced. The two major products, 20,24(OH)2D 3 and 20,25(OH)2D3, caused significantly greater inhibition of colony formation by SKMEL-188 melanoma cells than either 1,25(OH)2D3 or the parent 20(OH)D3, showing that CYP24A1 plays an activating, rather than an inactivating role on 20(OH)D3. © 2012 Elsevier Inc. All rights reserved.