The specific binding of [3H]threo-(+/-)-methylphenidate to membranes prepared from rat striatum was significantly reduced following either surgical lesions of the medial forebrain bundle or intracerebroventricular administration of 6-hydroxydopamine. The decrease in the density of [3H]threo-(+/-)-methylphenidate binding sites in striatum following chemical or surgical denervation was highly correlated with the decrease in [3H]dopamine uptake. In contrast, intracerebroventricular administration of 5,7-dihydroxytryptamine, AF64A, or chronic parenteral administration of reserpine did not alter either the number of apparent affinity of [3H]threo-(+/-)-methylphenidate binding sites. These data suggest that the specific binding sites for [3H]-threo-(+/-)-methylphenidate in striatum are localized to dopaminergic nerve terminals, and may be associated with the dopamine transport complex.