Methyl (2E,4R)-4-hydroxydec-2-enoate, methyl (2E,4S)-4-hydroxydec-2-enoate, and ethyl (±)-(2E)-4-hydroxy[4-2H]dec-2-enoate were chemically synthesized and incubated in the yeast Saccharomyces cerevisiae. Initial C-chain elongation of these substrates to C12 and, to a lesser extent, C14 fatty acids was observed, followed by γ-decanolactone formation. Metabolic conversion of methyl (2E,4R)-4-hydroxydec-2-enoate and methyl (2E,4S)-4-hydroxydec-2-enoate both led to (4R)-γ-decanolactone with >99% ee and 80% ee, respectively. Biotransformation of ethyl (±)-(2E)-4-hydroxy(4-2H)dec-2-enoate yielded (4R)-γ-[2H]decanolactone with 61% of the 2H label maintained and in 90% ee indicating a stereoinversion pathway. Electron-impact mass spectrometry analysis (Fig. 4) of 4-hydroxydecanoic acid indicated a partial C(4)→C(2) 2H shift. The formation of erythro-3,4-dihydroxydecanoic acid and erythro-3-hydroxy-γ-decanolactone from methyl (2E,4S)-4-hydroxydec-2-enoate supports a net inversion to (4R)-γ-decanolactone via 4-oxodecanoic acid. As postulated in a previous work, (2E,4S)-4-hydroxydec-2-enoic acid was shown to be a key intermediate during (4R)-γ-decanolactone formation via degradation of (3S,4S)-dihydroxy fatty acids and precursors by Saccharomyces cerevisiae.