Galactomyces reessii accomplishes the enzymatic transformation of beta-methylbutyric acid (isovaleric acid) to beta-hydroxy-beta-methylbutyric acid. The enzymatic basis for this bioconversion was evaluated by analyzing cell-free extracts of G. reessii for enzyme activities commonly associated with leucine catabolism. G. reessii extracts contained activities for acyl-CoA synthetase, acyl-CoA dehydrogenase, and enoyl-CoA hydratase, whereas beta-methylbutyric acid hydroxylase, alpha-ketoisocaproate oxygenase, and acyl-CoA oxidase (with isovaleryl-CoA as substrate) were not observed. Furthermore, beta-methylbutyric acid is initially activated to isovaleryl-CoA by acyl-CoA synthetase, dehydrogenated to methylcrotonyl-CoA by acyl-CoA dehydrogenase, hydrated to beta-hydroxy-beta-methylbutyric acid-CoA by enoyl-CoA hydratase, and hydrolyzed to beta-hydroxy-beta-methylbutyric acid in G. reessii extracts. Cell-free extracts converted both isovaleryl-CoA and methylcrotonyl-CoA into beta-hydroxy-beta-methylbutyric acid, thus demonstrating that beta-methylbutyric acid is part of the leucine catabolic pathway. The rate of beta-methylbutyric acid conversion to beta-hydroxy-beta-methylbutyric acid with cell-free extract was 0. 013 &mgr;mol beta-hydroxy-beta-methylbutyric acid (mg protein)-1 h-1, while the conversion rate of leucine was fivefold lower. With whole cells, the highest production rate [0.042 &mgr;mol beta-hydroxy-beta-methylbutyric acid (g cells)-1 h-1] was also observed with beta-methylbutyric acid. The results indicate that beta-methylbutyric acid is transformed to beta-hydroxy-beta-methylbutyric acid through the leucine catabolic pathway.