β-Adrenergic receptors in primary cultures of neonatal rat cardiac cells were identified with the radioligand [l25I]iodohydroxybenzylpindolol ([125I]IHYP). At the time of cell plating, a differ-ential attachment procedure was employed to separate myocardial (M) cells from fibroblast-like (F) cells. After 3-4 days, the cultures enriched in M cells were still more than 80% pure and the cultures enriched in F cells were more than 95% pure. For binding studies, confluent cell layers were nonenzy-matically detached as single cell suspensions. Both M and F cells contained a limited number of β-adrenergic receptors (M, 7600 % 2100 sites/cell; F, 9000 ± 2400 sites/cell) which had very high affinity (M, Kd= 88 % 33 PM; F, Kd= 71 % 23 pM) for [125I]IHYP. For each cell type, the binding sites were stereoselective for theI-isomers of agonists and antagonists. Further binding studies on the relative potency of β-agonists showed that the β-receptors in M cells could be subclassified as β1(isoproterenol > epinephrine = norepinephrine), whereas the β-receptors in F cells were more typical of β2(isoproterenol > epinephrine > norepinephrine). The order of potency of these catecholamines in stimulating the adenylate cyclase activity in M and F cells was consistent with the order of potency observed in binding studies. Practolol, a β1inhibitor, was 30 times more effective as a competitor of [125I]IHYP binding in M cells than in F cells. It thus appears that, whereas M and F cells share a similar number of receptors per cell and similar affinities for [125I]IHYP, the receptors in the two cell types nevertheless can be distinguished on the basis of their subclassification. These results also emphasize the importance of obtaining a homogeneous cell population for studies of the β-adrenergic receptor in cultured cardiac tissue.Circ Res 47: 41-48, 1980