It has been hypothesized, based on physiological evidence, that there is a greater proportion of β2-adrenergic receptors on the myocytes of the conduction system when compared with the working myocardium. The purpose of these studies was to examine β-adrenergic receptor subtype in the conduction system of the dog by using the technique of coverslip autoradiography. Scintillation studies of [125I]pindolol binding to ventricular sections demonstrated that binding was saturable (dissociation constant of 116 pM), had the correct order of potency for a 13-receptor, and was stereoselective. Both betaxolol (β1-selective) and ICI-118,551 (β2-selective) competition curves fit a two-site model in nonlinear curve-fitting analyses (78% β1-receptors). Autoradiographic studies determined that the myocytes of the sinoatrial node had approximately twice as many autoradiographic grains as the surrounding atrial myocytes. The myocytes of the atrioventricular bundle had a number of grains similar to the number in surrounding septal myocytes. Autoradiographic inhibition curves with betaxolol or ICI-118,551 demonstrated that both the sinoatrial node and the atrioventricular bundle had inhibition profiles similar to the surrounding myocytes (predominantly β1) but unlike the inhibition profiles of arterioles (predominantly β2). Calculations using the dissociation constants derived from the nonlinear curve-fitting analysis and the percent specific binding in the presence of 4×10−7M betaxolol or ICI-118,551 determined that the proportion of β1to β2-receptors was the same (70–80% β1) when comparing the sinoatrial node and the surrounding atrial myocytes. The atrioventricular bundle had a higher percentage of β1-receptors (95%) than the surrounding septal myocytes (79%) with betaxolol, but with ICI-118,551 the difference was not significant. Thus, by using techniques that can precisely quantify adrenergic receptors over myocytes of the conduction system, it was concluded that, whereas the myocytes of the sinoatrial node have approximately twice as many β-receptors as the surrounding atrial myocytes, the proportion of β1- to β2-receptors is the same.