Diastereomeric dihydrodiol metabolites of phenytoin, (5S)-5-[(3R,4R)-3,4-dihydroxy-1,5-cyclohexadien-1-yl]-5-phenylh yda ntoin, (S)-DHD, and (5R)-5-[(3R,4R)-3,4-dihydroxy-1,5-cyclohexadien-1-yl]-5-phenylh yda ntoin, (R)-DHD, have been resolved from each other and from urinary constituents with reversed-phase HPLC columns and acetonitrile-water gradients. Recoveries of DHD isomers from urine averaged 99.1% and it was demonstrated that known mixtures of DHD diastereomers added to blank urine were not altered by the assay procedures. The relative diastereomeric content of DHD was determined from integration of the chromatographic peaks. Assay of urine samples from patients on chronic phenytoin therapy and from volunteers indicated that both DHD isomers were present in all samples, and stereoselectivity favored the production of (S)-DHD.