TRH stimulation of prolactin release from GH3 cells is associated with loss of cellular Ca2+. Chlortetracycline (CTC), a fluorescent probe of Ca2+ in biological membranes, was previously employed to monitor indirectly changes in membrane Ca2+ in GH3 cells. Tetracaine, propranolol and trifluoperazine, agents that are known to displace Ca2+ from biological membranes, were utilized to demonstrate more rigorously that TRH affects cellular membrane Ca2+ in GH3 cells. Tetracaine (1 mM), propranolol (1 mM), and trifluoperazine (0.03 mM) inhibited basal and TRH-stimulated prolactin release, decreased cellular 45Ca2+ content and decreased cell-associated CTC fluorescence. Most importantly, these agents abolished the decrease in CTC fluorescence induced by TRH. These data suggest that tetracaine, propranolol and trifluoperazine displace membrane Ca2+ in intact GH3 cells and offer further evidence that TRH acts to mobilize cellular Ca2+ from a membrane-bound pool(s) during stimulation of GH3 cells.