Three methods for the analysis of the proportion of D-and L-pantoyl lactone in mixtures of these enantiomers are presented. They are: (a) the direct separation of the enantiomers by gas chromatography on a chiral stationary phase; (b) gas chromatographic separation of diastereoisomeric derivatives of pantoyl lactone; and (c) by calculation from circular dichroism measurements. The methods are applicable to the determination of the proportion of D-and L-pantoic acid in mixtures, after lactonization to pantoyl lactone. Results of the application of these methods to the determination of the stereochemistry oE (a) pantoyl lactone formed by ketopantoyl lactone reductases; and (b) pantoic acid formed by ketopantoic acid reductases of several microorganisms are presented. We previously demonstrated that KPL2 was stereospecifically reduced by [4A-3H]NADPH (1) to D-PL (2) by each of two highly purified forms of KPL reductase (3) from Saccharomyces cerevisiae (Fig. 1 This research was supported in part by the Veterans Administration. We express our appreciation to Dr. Phillip A. Hart for making available the spectropolarimeter and to Dr. John W. Porter for making available the gas chromatograph used in these studies. We also thank Dr. Arthur Kelman for providing specimens of E. herbicola, E. caratovora, and E. chrysanthemi, 2 Abbreviations used: KPL, ketopantoyl lactone (2-keto-4-hydroxy-3,3-dimethylbutyric acid-y-lactone); PL, pantoyl lactone (2,4-dihydroxy-3,3-dimethylbutyric acid-y-lactone); KPA, ketopantoic acid (2-keto-4hydroxy-3,3-dimethylbutyric acid); and PA, pantoic acid (2,4-dihydroxy-3,3-dimethylbutyric acid). [4A-3H]NADPH and [4B-3H]NADPH refer to the 4-R and 4-S forms of monotritiated NADPH, respectively. The R-S terminology conforms to the tentative rules of the IUPAC Commission on Nomenclature of Organic Chemistry (14). re and si faces refer to the nomenclature of two faces of a trigonal atom devised by Hanson ( 15). Glc-6-P, glucose 6-phosphate; glc, gas-liquid chromatography.