Abstract. The metabolic inhibitors antimycin A (2 μmol/l), dinitrophenol (0.5 mmol/l), and iodoacetate (6 mmol/l) were tested for their effects on hormone release, cAMP levels, and oxygen consumption in clonal strains of rat pituitary cells (GH3 cells). Basal release of growth hormone (GH) and prolactin (PRL) was reduced by all three inhibitors, and thyrotropin-releasing hormone (TRH) (l μmol/l) and K+ (50 mmol/l) stimulated hormone release were blocked. Trifluoperazine, a calmodulin antagonist, inhibited basal GH and PRL release at concentrations up to 30 μmol/l and stimulated above 50 μmol/l. The stimulatory effect of 80 μmol/l trifluoperazine on basal hormone release was eliminated by antimycin A, dinitrophenol, and iodoacetate, whereas the inhibitory effect of antimycin A, dinitrophenol and iodoacetate on basal hormone was not affected by 30 μmol/l trifluoperazine. None of the inhibitors had any effect on the level of cellular cAMP (i.e. intracellular plus extracellular). Oxygen consumption of GH3 cells was blocked by antimycin A, reduced by 25% by iodoacetate and increased by about 100% by dinitrophenol. In contrast, hormone secretion stimulated by TRH and K+ was not accompanied by any measurable alteration in oxygen consumption. Trifluoperazine (⩾ 80 μmol/l) reduced the basal oxygen consumption and blocked the stimulatory effect of dinitrophenol on oxygen consumption. In conclusion, inhibition of the energy generation of GH and PRL-producing cells severely affects the action of secretagogues, although stimulated hormone secretion may not be accompanied by any measurable increase in oygen consumption. The cellular energy supporting hormone secretion is mostly generated via oxidative phosphorylation.