With agonist stimulation, cardiac β2-adrenergic receptors (β2ARs) are downregulated to a much greater extent than are β1ARs. It has been hypothesized that this effect is due to sympathetic innervation inhibiting the downregulation of β1ARs. To test this hypothesis, the technique of coverslip autoradiography was used to localize and quantify β1AR and β2AR subtypes in tissue compartments of the heart in rats subjected to sympathetic denervation by two intravenous injections of 6-hydroxydopamine (50 mg/kg per dose). After denervation, the rats were infused with l-isoproterenol (400 μg-kg−1 - h−1 for 7 days) or vehicle (0.001N HCI) by implantation of osmotic minipumps. Sections were incubated with 70 pmol/L of the βAR antagonist [125I]iodocyanopindolol (ICYP) alone or in the presence of 5 μmol/L dl-propranolol or 5×10−7 mol/L CGP 20712A (a β1AR antagonist). Binding of ICYP to sections of rat hearts was saturable and stereoselective and was displaced by βAR agonists with the rank order of potency expected for βARs. There was an 89% reduction in catecholamine concentration in rat ventricles after 1 week of 6-hydroxydopamine treatment, before implantation of the minipumps. Chronic infusion of isoproterenol induced significant downregulation (63% to 74%) of β2ARs in atrial and ventricular myocytes, coronary arterioles, and connective tissue but no change in β1ARs in these regions in rats with intact sympathetic innervation. Similar changes were seen in denervated animals. There was a marked reduction in β2ARs but small insignificant decreases in β1ARs, despite the fact that in the denervated animals there was upregulation of β1ARs in atrial and ventricular myocytes (≈25%). Our study suggests that β1ARs in the heart are not significantly downregulated by chronic agonist exposure and that this is unrelated to sympathetic innervation. The underlying mechanism of preferential regulation of βAR subtypes remains to be elucidated but may be related to differences in the molecular structure between β1ARs and β2ARs.