It has been suggested that there is a preferential coupling in heart muscle between the inhibitory G protein (G_i) and the ₂-subtype of the -adrenergic receptor (-AR), since pertussis toxin (which inactivates G_i) reveals latent ₂-ARs in rat and mouse myocytes. We have previously shown that guinea pigs treated with norepinephrine (NE) for 7 days have myocytes that are desensitized to -AR-agonist stimulation, and that pertussis toxin restores these responses. The purpose of the present investigation was to determine whether pertussis toxin specifically upregulated ₂-ARs in myocytes from NE-treated guinea pigs. The sole -AR subtype in control guinea pig myocytes was confirmed as ₁-AR by radioligand binding, single-cell autoradiography, and concentration-response curves to isoproterenol in contracting myocytes. In contrast, a minor pool of ₂-ARs was observed in rat myocytes by use of the same methods. NE treatment decreased the maximum isoproterenol response (relative to high Ca²⁺) from 0.89 ± 0.06 to 0.58 ± 0.08 (n = 7, P < 0.01) and the pD₂ (log EC₅₀) from 8.8 ± 0.2 to 7.5 ± 0.2 (n = 7, P < 0.01). Pertussis toxin treatment increased the isoproterenol-to-Ca²⁺ ratio to 0.88 ± 0.04 (n = 6, P < 0.05) and the pD₂ to 8.6 ± 0.3 (P < 0.01). This was not mediated by increases in either number or function of ₂-ARs. G_i is therefore able to modulate ₁-AR responses in guinea pig myocytes.