The intermolecular primary deuterium isotope effects on the individual C-H bond cleavage steps involved in dihydroceramide Delta (4) desaturation have been determined for the first time by incubating rat liver microsomes with 1:1 mixtures of nonlabeled substrate and the appropriate regiospecifically dideutelatred analogue. Analysis of the enzymatic products via gas chromatography coupled to mass spectrometry showed that the introduction of the (E) double bond between C-4 and C-5 occurs in two discrete steps: cleavage of the C-4-H bond was found to be very sensitive to isotopic substitution (k(H)/k(D) = 8.0 +/-0 8). while a negligible isotope effect (k(H)/k(D) = 1.02 +/-0.07) was observed for the C-5-H bond-breaking step. According to a mechanistic model that we have previously proposed, these results suggest that initial oxidation for this desaturation reaction occurs at C-4. This finding correlates nicely with the observation that 4-hydroxylated products are produced from a similar substrate by a closely related oxidative enzyme in yeast.