In this study, we hypothesized that a reduction in beta-adrenergic receptor number or a decrease in functional coupling of the receptor to the adenylate cyclase system may be responsible for the blunted inotropic response to isoproterenol observed in fetal sheep exposed to high altitude (3,820 m) from 30 to 138-142 days gestation. We measured the contractile response to increasing doses of isoproterenol and forskolin in papillary muscles from both ventricles, estimated beta-adrenergic receptor density (Bmax) and ligand affinity (Kd) using [125I]iodocyanopindolol, and measured adenosine 3',5'-cyclic monophosphate (cAMP) levels before and after maximally stimulating doses of isoproterenol and forskolin. Left ventricular wet weight was unchanged, but right ventricular weight was 20% lower than controls. At the highest concentration of isoproterenol (10 microM), maximum active tension was 32 and 20% lower than controls in hypoxemic left and right ventricles, respectively. The contractile response to forskolin was severely attenuated in both hypoxemic ventricles. Bmax was unchanged in the left ventricle, but increased by 55% in the hypoxemic right ventricle. Kd was not different from controls in either ventricle. Basal cAMP levels were not different from controls, but isoproterenol-stimulated and forskolin-stimulated cAMP levels were 1.4- to 2-fold higher than controls in both hypoxemic ventricles. The results suggest mechanisms downstream from cAMP in the beta-adrenergic receptor pathway are responsible for the attenuated contractile responses to isoproterenol.