Abstract The novel specificity of a new R-stereospecific 2-hydroxyisocaproate dehydrogenase R-HicDH) from Lactobacillus casei is reported. Such enzymes are of practical importance for the preparation of chiral 2-hydroxy acids of value as synthons in asymmetric synthesis. While there are examples of S-stereospecific enzymes with better tolerance for different substrate structures, the application of R-specific enzymes has until now been hampered by their narrow substrate specificity. R-HicDH, which has been cloned and overexpressed in Escherichia coli, overcomes this limitation, thus permitting the enzymatic synthesis of a wide range of enantiomerically pure R-2-hydroxy acids. The catalytic properties of this enzyme with a wide range of 2-hydroxy and 2-oxocarboxylic acid substrates are reported. The enzyme has been shown to have a broad substrate specificity, and efficient conversions in both reductive and oxidative directions are readily achievable. Furthermore, water-miscible organic solvents are tolerated as cosolvents at concentrations from 10% to 35%. The synthetic applicability of R-HicDH is demonstrated by the preparation of enantiomerically pure R-2-hydroxy-4-methylpentanoic acid in 88% yield, using formate dehydrogenase recycling of the NADH coenzyme.