Washed cells of Rhodococcus erythropolis IFO 12540 were found to convert only the l-(+)-isomer of pantoyl lactone to the d-(-)-isomer in a racemic mixture of pantoyl lactone. Under suitable reaction conditions, the amount of d-(-)-pantoyl lactone synthesized was 18.2 mg/ml (94.4% enantiomer excess; molar yield, 90.5%). This conversion was suggested to proceed through the following successive reactions: first, the enzymatic oxidation of l-(+)-pantoyl lactone to ketopantoyl lactone; second, the rapid and spontaneous hydrolysis of the ketopantoyl lactone to ketopantoic acid; and then, the enzymatic reduction of the ketopantoic acid to d-(-)-pantoic acid. After the reaction d-(-)-pantoic acid could be lactonized by means of acid treatment. During the conversion, the d-(-)-isomer, which was initially present in the reaction mixture, did not undergo any modification.