We have previously found that the D₅ dopamine receptor couples to a G-protein other than G_sα, and could be involved in signaling pathways other than regulation of adenylyl cyclase. To describe interactions of the D₅ receptor with cellular effectors, we used GH₄C₁ cells transfected with cDNA for the human D₅ receptor. Thyrotropin-releasing hormone (TRH, 100 nM) stimulated accumulation of inositol phosphates (IPs) fivefold in D₅GH₄C₁ cells. Dopamine (DA, 10 µM) inhibited TRH-stimulated IP values by 29%; at higher concentrations (100 µM), maximal inhibition of 61% was observed. The D₅ agonist SKF R-38393 (10 µM) mimicked this effect (28% inhibition). SCH 23390, a D₅ antagonist, blocked the inhibition caused by both DA and SKF R-38393. Spiperone, a D₂ receptor antagonist, did not block the inhibition. The D₂ agonist (±)-2-(N-phenylethyl-N-propyl)amino-5-hydroxytetralin (PPHT) did not inhibit TRH-stimulated IP production, nor did it augment the effect of D₅ agonists. The DA-mediated suppression of IP levels was not sensitive to pertussis toxin; cholera toxin blocked both TRH stimulation and DA suppression of IP accumulation in response to 100 nM TRH. Neither dibutyryl cAMP nor forskolin lowered IP formation in response to TRH. Phorbol ester decreased TRH-stimulated IP accumulation in D₅GH₄C₁ cells; however, an inhibitor of protein kinase C (PKC) did not block the effect of DA.