The goal of the current study was to determine which of the D2-like receptors (D2, D3 or D4) are involved in autoreceptor regulation of dopamine synthesis. We have derived a model system utilizing a mouse mesencephalic cell line, MN9D, which both synthesizes and releases dopamine, to characterize the modulation of tyrosine hydroxylase activity, the rate limiting enzyme in the conversion of tyrosine to dopamine, by the D2-like receptors. Previously, we have shown that stimulation of D2 and D3, but not D4, dopamine receptors transfected into MN9D cells inhibited the release of dopamine. In the current study, we show that quinpirole stimulation of transfected D2 and D3, but not D4, dopamine receptors inhibited K+-stimulated tyrosine hydroxylase activity in a pertussis toxin-sensitive manner, strongly suggesting G-protein coupling as a mechanistic pathway. The D2 receptor effect could be maintained for at least 60 min, whereas the D3 receptor effect desensitized. Treatment with 10 microM forskolin, which raises cyclic AMP levels or with 100 nM okadaic acid, a potent phosphatase inhibitor, had no effect on the D2-or D3-mediated inhibition, suggesting that these effects may be independent of both cyclic AMP- and okadaic acid-sensitive phosphatase activity. Taken together, these data confirm the hypothesis that dopamine D2 and D3 receptors can perform dual roles in autoreceptor regulation.