The intermolecular primary deuterium isotope effects on the individual C-H bond cleavage steps involved in linoleic acid biosynthesis were determined using a suitably transformed strain of Saccharomyces cerevisiae containing a functional oleate Delta(12) desaturase from Arabidopsis thaliana. Mass spectral analysis of the methyl 7-thialinoleate fraction obtained from competition experiments involving methyl 7-thiastearate, methyl [12,12-H-2(2)]-7-thiastearate and methyl [13,13-H-2(2)]-7-thiastearate showed that cleavage of the C-12-H bond is very sensitive to isotopic substitution (k(H)/k(D) = 7.3 +/- 0.4) while a negligible isotope effect (k(H)/k(D) = 1.05 +/- 0.04) was observed for the C-13-H bond breaking step. This result strongly suggests that the site of initial oxidation for Delta(12) desaturation is at C-12. The possible relationship between castor oleate 12-hydroxylase and microsomal Delta(12) oleate desaturases is discussed in the context of a common mechanistic paradigm. Our methodology may be also be useful in deciphering the cryptoregiochemistry of other desaturase systems.