The stereochemistry of dehydrogenation of the primary carbinol group of D-galactose by D-galactose oxidase has been determined. Using D-galactose-6-d and methyl β-D-galactopyranoside-6-d, it has been established that the reaction involves removal of the pro-S 6-hydrogen atom. This conclusion is based on product analysis, and on the relative impact of the deuterium isotope effect on oxidation rates of substrates having different R:S deuteration patterns. The absolute configurations at C-6 of these substrates have been determined by selective chemical transformations to products of known configuration. The rotational conformation of the 6-carbinol group of D-galactose and its possible relationship to the specificity of the enzyme are discussed, as well as the stereochemistry of reductive deuteration of aldehydo sugar derivatives.