To investigate the stereoselective distribution of methylphenidate (MPD) enantiomers in rats, the concentrations of each enantiomer were determined in plasma and brain regions (cerebellum, striatum, basal forebrain, brain stem, and cortex) after iv administration of racemic MPD and its individual enantiomers. The concentrations of MPD enantiomers in each brain region reached pseudo-steady state within 10 min after iv administration of racemic MPD (2 mg/kg dose). The influx clearances for MPD calculated from Kpapp values in each brain region were not significantly different between MPD enantiomers and between the five brain regions. The mean Kpapp values for (+)-MPD in the striatum at 120 and 240 min after administration of racemic MPD were 10.1 and 10.5, respectively, and these values at each time were significantly larger than the Kpapp values (7.5 and 7.0, respectively) for the (-)-isomer (P < 0.01). The Kpapp value for (+)-MPD in the striatum decreased by coadministration of mazindol as an inhibition of both dopamine and norepinephrine reuptake, but it was not changed by desipramine as a norepinephrine reuptake inhibitor. These results suggest that (+)-MPD was bound specifically to the dopamine reuptake site in the striatum.