Prolonged exposure of the skin to UV radiation causes pre-vitamin D3, the initial photoproduct formed by opening of the B ring of 7-dehydrocholesterol, to undergo a second photochemical reaction where the B-ring is reformed giving lumisterol 3 (L3), a stereoisomer of 7-dehydrocholesterol. L3 was believed to be an inactive photoproduct of excessive UV radiation whose formation prevents excessive vitamin D production. Recently, we reported that L3 is present in serum and that CYP11A1 can act on L3 producing monohydroxy- and dihydroxy-metabolites including 24(OH)L3, which exhibit biological activity on skin cells comparable to 1,25(OH)2D3. Since human CYP27A1 can hydroxylate 7-dehydrocholesterol we tested its ability to hydroxylate L3. It metabolised L3 to 3 major products identified by NMR as 25-hydroxylumisterol and the two C25 enantiomers of 27-hydroxylumisterol, (25R)-27-hydroxylumisterol and (25S)-27-hydroxylumisterol. These three major products were also seen when mouse liver mitochondria containing CYP27A1 were incubated with L3. The requirement for CYP27A1 for their formation by mitochondria was confirmed by the inhibition of their synthesis by 5β-cholestane-3α,7α,12α-triol, an intermediate in bile acid synthesis which serves as an efficient competitive substrate for CYP27A1. The kinetics of L3 metabolism by purified human CYP27A1 were determined with substrate incorporated into the membrane of phospholipid vesicles which revealed a very high kcat (76 mol product/min/mol CYP27A1) and a catalytic efficiency (kcat/Km) that was 260-fold higher than for vitamin D under identical conditions. The CYP27A1-derived hydroxy-derivatives inhibited the proliferation of cultured human melanoma cells and colony formation in soft agar with IC50 values in the nM range. Thus, L3 is metabolised efficiently by CYP27A1 with hydroxylation occurring at C25 or C27. The products are potent in their ability to inhibit melanoma cell proliferation and may represent a new class of hormones dependent on UV radiation for their synthesis.