A novel enzymatic process for the optical resolution of racemic pantolactone through the stereospecific hydrolysis of D-pantolactone by lactonohydrolase of Fusarium oxysporum is described. F, oxysporum cells were found to catalyze the stereoselective hydrolysis of the D-enantiomer of racemic pantolactone. With 135 g/l DL-pantolactone as the substrate, 41% was hydrolyzed and pantoic acid with an optical purity of 90% enantiomeric excess (for D-pantoic acid) was formed.