The determination of the absolute stereochemistry of hydroxy fatty acid methyl esters as their (S)-ibuprofen esters is possible via standard gas chromatographic techniques. Analyses of various racemic and nonracemic standards and mixtures from enzymic oxidation show excellent resolution of the resultant diastereomers, with the (S,S)-diastereomers eluting first in all cases studied. The stereochemistry of the oxidation of dodecanoic acid by P450BM3, which has not been previously reported, was determined by this method and indicated a preference for (R)-hydroxylation. The sensitivity of this technique allows the analysis of very small quantities of product, which has revealed that the oxidation of dodecanoic and hexadecanoic acids by the T268A mutant of P450BM3 display the same stereochemical efficiency and produce (R)-hydroxy fatty acids in the same manner as wildtype P450BM3, despite the poor coupling efficiency of these substrates. This stereochemistry implies that hydroxylation catalysed by the T268A mutant of P450BM3 occurs through residual levels of the normal hydroxylating species.