The human heart contains at least four distinct β-adrenoceptor subtypes, three of which have been cloned. However, the binding properties of β-blockers to the different β-adrenoceptor subpopulations are not yet thoroughly characterized. Human β1-, β2-, and β3-adrenoceptors were expressed in COS-7 cells and [125I]iodocyanopindolol saturation binding, and competition experiments with commonly used β-blockers were performed in the respective membrane preparations. Atenolol and metoprolol were about fivefold selective for β1- versus β2- and β3-adrenoceptors. Bisoprolol was ∼15-fold selective for β1- versus β2- and ∼31-fold selective for β1- versus β3-adrenoceptors. Carvedilol was nonselective for any β-adrenoceptor subtype. We conclude that the β1-selectivities of atenolol, metoprolol, and bisoprolol are lower in COS cell membranes compared with previous investigations performed in native membranes. All β-blockers investigated bind to β3-adrenoceptors. Differential binding properties to β3-adrenoceptors might imply different responses as to body weight, cardiac contractility, heart rate, and growth regulation. This might imply differential indications for the drugs investigated.