An enantioselective gas chromatographic quantitation assay was developed for the enantiomers of dl-threo-methylphenidate in plasma and urine. dl-threo-Methylphenidate and the internal standard were acylated with N-heptafluorobutyryl-1-prolylchloride under Schotten-Baumann conditions prior to gas chromatographic separation on achiral mixed stationary phases. The derivatives were detected by means of a nitrogen-phosphorus detector. Linear and reproducible calibration curves were obtained over the concentration ranges 0.43-43.25 and 2.16-216.25 ng/ml enantiomer in plasma or urine, respectively. This enantioselective gas chromatographic quantitation assay was applied in a single oral dose disposition study of dl-threo-methylphenidate in a healthy adult volunteer. Stereoselective differences were observed in the plasma concentration-time profiles and cumulative urinary excretion profiles following oral doses of 20 and 40 mg of dl-threo-methylphenidate hydrochloride. Only d-threo-methylphenidate was detectable in plasma after 4 h.