Incubation of valproic acid with rat liver microsomes led to the formation of 3-, 4- and 5-hydroxy-valproic acid. The latter two metabolites, which have been characterized previously from in vivo studies, may be regarded as products of fatty acid omega-1 and omega hydroxylation, respectively. 3-Hydroxy-valproic acid, however, had been thought to derive from the beta-oxidation pathway in mitochondria. Conversion of valproic acid to all three metabolites in microsomes required NADPH (NADH was less effective), utilized molecular oxygen, was suppressed by inhibitors of cytochrome P-450 and was stimulated (notably at C-3 and C-4) by phenobarbital pretreatment of the rats. It is concluded that rat liver microsomal cytochrome P-450 catalyzes omega-2 hydroxylation of valproic acid, a reaction not detected previously with fatty acids in mammalian systems, and that the product, 3-hydroxy-valproic acid, should not be used to assess in vivo metabolism of valproate via the beta-oxidation pathway.