In order to investigate the possibility that there may be two conformationally distinct dopamine D1 binding sites, the effect of lysine-modifying agents on striatal dopamine D1 receptors was investigated. Treatment with the distilbene derivative, 4,4'-diisothiocyanostilbene-2,2'-disulfonate, (DIDS), resulted in an irreversible D1 receptor inactivation that was associated with a 70% loss of binding sites. The remaining DIDS-insensitive sites displayed both a decreased affinity (approximately 5 fold) for the D1 antagonist SCH-23390 and an enhanced affinity of dopaminergic agonists (approximately 10 fold) for the agonist high-affinity form of the receptor. Pretreatment with Gpp(NH)p, a non-hydrolysable guanine nucleotide, prevented the formation of the agonist high-affinity form, indicating that these sites are G-protein-linked. Prior occupancy of D1 receptors with dopaminergic agonists and antagonists afforded no protection against DIDS inactivation, suggesting that a site outside the ligand binding subunit of the D1 receptor was modified. Taken together, these data suggest that [3H]SCH-23390 labels two conformationally distinct populations of dopamine D1 receptors.