Regulation of the rate-limiting step in dopamine (DA) synthesis was estimated in striatal synaptosomes by measuring the rate of hydroxylation of L-4-[3H]phenylalanine, a substrate of tyrosine hydroxylase (TH). DA inhibited hydroxylation with an IC50 of 0.2 microM. The concentration-response curve of DA-induced inhibition was not affected by the presence of 1 microM chlorpromazine, a phenothiazine DA antagonist. Sulpiride and haloperidol, DA antagonists of the benzamide and butyrophenone classes respectively, also failed to alter the inhibition of substrate hydroxylation by 1 microM DA, even at concentrations up to 10 microM. In contrast, a parallel 15 fold shift to the right in the concentration-response curve of DA-induced inhibition of hydroxylation was obtained when 10 microM nomifensine, a competitive DA uptake inhibitor, was added. Even in the presence of nomifensine, 1 microM chlorpromazine had no effect on the DA concentration-response curve. The addition of DMPH4, an artificial cofactor for TH, completely blocked DA-induced inhibition of enzymatic activity. These data suggest that direct autoreceptor control of synaptosomal TH activity does not exist in vitro, and that DA-induced inhibition of TH occurs subsequent to reuptake via classical feedback inhibition, presumably by competitive displacement of the necessary endogenous cofactor.