Two major classes of dopamine receptors, called D-1 receptors and D-2 receptors, have been identified. [alpha-3H]Flupenthixol has been used as a radioligand for the study of D-1 receptors, which are thought to act through stimulation of adenylate cyclase activity. Previous studies in our laboratory have shown that the D-2 receptors in rat caudate labeled by [3H]spiroperidol also have a high affinity for alpha-flupenthixol. The present experiments show that although Scatchard analysis of the binding of [alpha-3H]flupenthixol is consistent with the presence of a homogeneous population of receptors, subpopulations of these sites can be distinguished by their differing affinities for spiroperidol, which has a Kd for D-1 receptors of about 0.3 microM and a Kd for D-2 receptors of approximately 50 pM. The number of D-1 receptors in rat striatum is approximately 4 times the number of D-2 receptors. D-1 receptors can be studied by including 10 nM spiroperidol in assays carried out with [alpha-3H]flupenthixol, thus blocking the binding of [alpha-3H]flupenthixol to D-2 receptors. The affinity of these receptors for dopamine is decreased by GTP, as has been observed in studies of other receptors whose effects are mediated through changes in adenylate cyclase activity. In the presence of spiroperidol, the Hill coefficients determined from dose-response curves of the inhibition of the binding of [alpha-3H]flupenthixol by antagonists or by agonists in the presence of GTP suggest that the binding reaction obeys simple Michaelis-Menten kinetics for a single class of binding sites.