We have investigated the intracellular mechanisms underlying thyrotropin-releasing hormone (TRH)-mediated [3H]dopamine ([3H]DA) release from dispersed rat tuberoinfundibular dopaminergic (TIDA) neurons. The specific binding of [3H]Me-TRH to these cells is characterized by a single, high-affinity binding site (Kd = 1.2 nM) with a Bmax value of 178 fmol/mg protein. Thyrotropin-releasing hormone markedly increased [3H]DA release and intracellular free calcium concentration ([Ca2+]i) in TIDA neurons, and its effect was abolished by treatment with EGTA (5 mM) or chlordiazepoxide, a specific TRH receptor antagonist (10 microM). Furthermore, to examine the involvement of protein kinase C on [3H]DA release, we investigated the effect of phorbol myristate acetate (PMA), which is known to activate protein kinase C directly. Phorbol myristate acetate induced a significant increase in [3H]DA release in a concentration-dependent manner. Treatment with TRH (1 microM) plus PMA (100 nM) resulted in an additive increase in [3H]DA release. Thyrotropin-releasing hormone (1 microM) still increased [3H]DA release even after preincubation with PMA (500 nM) for 24 h, but PMA (100 nM) did not under the same conditions. These results suggest that TRH may induce DA release in dispersed rat TIDA cells by increasing calcium influx and activating the protein kinase C system.