Rats were administered threo-dl-methylphenidate (MPH) i.v., i.p. and p.o. A gas chromatography-mass spectrometry method incorporating deuterated internal standards was used to quantify MPH and the metabolite threo-dl-p-hydroxymethylphenidate (HOMPH). Accumulation and decay of MPH were evaluated in serum and brain to assess whether serum MPH correlated with brain content. HOMPH was quantified to appraise its contribution to the central action of MPH. MPH accumulated in the brain at a concentration of 3.75 microgram/g tissue within 1 min after i.v. injection of 1 mg/kg, and averaged 8 times the concentration in serum over the first 30 min. The steady-state volume of distribution was 1.47 liters/kg. The decay of MPH from brain and serum occurred biphasically with postdistribution (beta) T1/2 values of 20 and 50 min, respectively. After p.o. administration (1 mg/kg), brain MPH concentrations also exceeded those in serum. HOMPH was detected only in liver and kidney after 1 mg/kg MPH (i.v. and p.o.). Thirty minutes after 20 mg/kg of MPH (i.p.), MPH accumulated in kidney greater than lung greater than brain greater than heart greater than liver. Brain MPH concentration was 900 times that of HOMPH (0.02 microgram/g) after this dose. In liver, conjugated HOMPH was present (12.1 microgram/g). After i.v. administration of HOMPH, less localization occurred in brain than occurred for MPH. These data suggest that the central effects of MPH do not depend upon conversion to HOMPH.