The pharmacology of the enantiomers of threo-methylphenidate (MPH) was evaluated in the rat to assess the relative contribution of each isomer to central and peripheral actions of the racemic drug. Fractional recrystallization of binaphthyl phosphate salts of dl-threo-MPH allowed resolution of d-threo-MPH and 92% enrichment of l-threo-MPH. The enantiomeric disposition was monitored using gas chromatographic separation of trifluoroacetylprolyl diastereomeric derivatives. The activity of the d-isomer was greater than the l-isomer in the induction of locomotor activity and the inhibition of tritiated dopamine and l-norepinephrine uptake into striatal and hypothalamic synaptosomes, respectively. Neither isomer produced a significant change in the spontaneous release of tritiated catecholamines from synaptosomes. Destruction of catecholaminergic neurons by 6-hydroxydopamine pretreatment attenuated the locomotor response to d-threo-MPH, indicating the involvement of catecholaminergic neural pathways in the locomotor response. Only the d-enantiomer significantly potentiated the pressor responses to i.v. l-norepinephrine. Receptor site stereoselectively for threo- vs. erythro-MPH is discussed in terms of isomer conformational preferences. These results suggest that synaptic inhibition of catecholamine uptake by d-threo-MPH may be involved fundamentally in behavioral and pressor effects of the racemic drug.