We have demonstrated that the rat pituitary tumor cell line GH3 has a carrier-mediated active transport system for the beta-adrenergic antagonist dihydroalprenolol (DHA). Transport of DHA in GH3 was saturable, with an apparent Km of 1.4 microM, was temperature and pH dependent, and was inhibited by the ionophore monensin and the amine transport inhibitor reserpine. Propranolol competed for DHA transport, but not in a stereoselective fashion. The tricyclic antidepressant imipramine also competed for DHA transport, but catecholamines or serotonin did not. This amine transport system in GH3 cells appeared to be identical to the one we recently described in several other cell types; however, analysis in those cells was complicated by the fact that they contain beta-adrenergic receptors which bind beta-adrenergic ligands. In this report we show that GH3 cells do not possess detectable beta-adrenergic receptors, based on their inability to bind the partial agonist CGP-12177, their inability to bind nanomolar concentrations of DHA in a saturable, stereospecific manner, and their failure to produce cAMP in response to stimulation by beta-adrenergic agonists. Characterization of the amine transport system in GH3 cells clearly distinguishes it from receptor-mediated phenomena and should facilitate our efforts to fully understand its mechanism and significance.