Superfusion with dopamine (0.1 microM-10 mM) evokes calcium-dependent [3H]acetylcholine release from rabbit retina labeled in vitro with [3H]choline. This effect is antagonized by the D-1 dopamine receptor antagonist SCH 23390. Activation or blockade of D-2 dopamine, alpha-2 or beta receptors did not stimulate or attenuate the release of [3H]acetylcholine from rabbit retina. Dopamine receptor agonists evoke the release of [3H]acetylcholine with the following order of potency: apomorphine greater than or equal to SKF(R)82526 greater than SKF 85174 greater than SKF(R)38393 greater than or equal to pergolide greater than or equal to dopamine (EC50 = 4.5 microM) greater than SKF(S)82526 greater than or equal to SKF(S)38393. Dopamine receptor antagonists inhibited the dopamine-evoked release of [3H]acetylcholine: SCH 23390 (IC50 = 1 nM) greater than (+)-butaclamol greater than or equal to cis-flupenthixol greater than fluphenazine greater than perphenazine greater than trans-flupenthixol greater than R-sulpiride. The potencies of dopamine receptor agonists and antagonists at the dopamine receptor mediating [3H]acetylcholine release is characteristic of the D-1 dopamine receptor. These potencies were correlated with the potencies of dopamine receptor agonists and antagonists at the D-1 dopamine receptor in rabbit retina as labeled by [3H]SCH 23390, or as determined by adenylate cyclase activity. [3H]SCH 23390 binding in rabbit retinal membranes was stable, saturable and reversible. Scatchard analysis of [3H]SCH 23390 saturation data revealed a single high affinity binding site (Kd = 0.175 +/- 0.002 nM) with a maximum binding of 482 +/- 12 fmol/mg of protein. The potencies of dopamine receptor agonists to stimulate [3H]acetylcholine release were correlated with their potencies to stimulate adenylate cyclase (r = 0.784, P less than .05, n = 7) and with their affinities at [3H]SCH 23390 binding sites (r = 0.755, P greater than .05, n = 8). The potencies of antagonists to inhibit dopamine-evoked [3H]acetylcholine release were correlated with their potencies to inhibit the dopamine-stimulated adenylate cyclase (r = 0.759, P less than .05, n = 5) and with their affinities at [3H]SCH 23390 binding sites (r = 0.998, P less than .01, n = 7). We conclude that in rabbit retina dopamine evokes calcium-dependent [3H]acetylcholine release through activation of a site with the pharmacological characteristics of a D-1 dopamine receptor.