The effects of alkylating agent N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) on the binding of [3H]7-OH-DPAT, a ligand for the D3 dopamine receptor, were assessed in ventral striatal (n. accumbens and olf. tubercle) membranes of adult, male Sprague-Dawley rats. [3H]Spiperone binding to D2-like receptors in striatal membranes was also assayed as a positive control. In vitro, EEDQ was equipotent in inactivating [3H]7-OH-DPAT- and [3H]spiperone-labeled binding sites. In vitro, [3H]spiperone binding was rapidly eliminated in a dose-dependent manner following EEDQ administration. In contrast, [3H]7-OH-DPAT binding was not significantly altered by any dose of the alkylating agent at any time point examined. Depletion of endogenous catecholamines with alpha-methyltyrosine and reserpine revealed a second, higher affinity binding site for [3H]7-OH-DPAT. Administration of EEDQ in catecholamine-depleted animals reduced [3H]7-OH-DPAT labels two distinct populations of binding binding sites in rat brain membranes, only one of which is susceptible to inactivation by EEDQ. These sites may represent high and low affinity states of the D3 receptor. In addition, this discovery may provide a useful method for examining the function of some D3 receptors in brain independent of other monoaminergic systems.