The mechanism of formation of the in-chain, unsaturated fatty acid metabolite, Δ 3 -valproic acid (Δ 3 -VPA) by rat liver microsomes was examined. Microsomal rates of formation of Δ 3 -VPA were below quantifiable limits in reactions catalyzed by control female rat liver microsomes, but were induced more than 20-fold following pretreatment with triacetyloleandomycin and pregnenolone-16α-carbonitrile. Microsomal incubations conducted with 3-hydroxy-VPA or [2- 2 H 1 ]VPA demonstrated that Δ 3 -VPA did not arise by dehydration of preformed alcohol nor was it reversibly isomerized to Δ 2 -VPA. CYP3A1 expression was optimized in the baculovirus expression vector system, and infected insect cell membranes which were supplemented with P450 reductase catalyzed formation of 3-OH-, 4-OH-, 5-OH-, Δ 3 -, and Δ 4 -VPA in ratios of 160:35:6:3:1. Intramolecular deuterium isotope effects on metabolite formation, determined with cDNA-expressed CYP3A1 and either [3,3- 2 H 2 ]VPA or [4,4- 2 H 2 ]VPA, yieldedk H /k D values for Δ 3 -VPA of 2.00 +/- 0.06 and 2.36 +/- 0.08, respectively. These values were significantly lower than the isotope effects observed in the same incubations for 3-OH-VPA formation from 3,3-D 2 -VPA (k H /k D = 6.04 +/- 0.08), or for 4-OH- and Δ 4 -VPA formation from 4,4-D 2 -VPA (k H /k D > 5). Collectively, these data demonstrate the existence of a microsomal P450-dependent in-chain fatty acid desaturase system distinct from the well-documented cytochromeb 5 -linked CoA desaturases and suggest further that CYP3A1-dependent formation of Δ 3 -VPA arises via nonselective, initial hydrogen atom abstraction from either the C-3 or the C-4 position.