The binding of dopaminergic agonist and antagonist radioligands was investigated in rat brain regions. A 30-min preincubation of homogenates of caudate nucleus or mesolimbic brain regions at 37 degrees induced a several-fold increase in the stereospecific binding of [3H]-dopamine or [3H]apomorphine to the subsequently washed particulate fraction, whereas it induced a slight decrease in [3H]spiroperidol binding. Stereospecific 3H-agonist binding was not observed in brain regions devoid of dopaminergic innervation. Guanosyl nucleotides, EDTA or ethyleneglycol-bis-(beta-amino-ethyl ether) N,N'-tetraacetate (EGTA), included in the preincubation buffer, antagonized the stimulation of 3H-agonist binding. The stereospecific binding sites of 3H-agonists were saturable with equilibrium dissociation constants (Kd) of 1-2 nM. High-affinity binding was pH dependent, with different pH optima for each radioligand. Several dopamine receptor agonists and antagonists were potent inhibitors of stereospecific [3H]dopamine binding, whereas l-ascorbic acid was inactive at concentrations as high as 1.0 mM. The stereospecific binding of [3H]apomorphine or [3H]spiroperidol was also unaffected by ascorbic acid. The nonspecific (d-butaclamol-insensitive) portion of 3H-agonist binding was weakly inhibited by concentrations of ascorbic acid higher than 0.01 mM. This effect was also observed in the cerebellum and spinal cord, where none of the 3H-agonist binding was stereospecific. It is concluded that the portion of 3H-agonist or 3H-antagonist radioligand binding which is related to dopamine receptors is unaffected by ascorbic acid.