Antagonists of β-adrenergic receptors (β-ARs) have become a main therapeutic regimen for the treatment of heart failure even though the mechanisms of their beneficial effects are still poorly understood. Here, we used fluorescent resonance energy transfer–based (FRET-based) approaches to directly monitor activation of the β₁-AR and downstream signaling. While the commonly used β-AR antagonists metoprolol, bisoprolol, and carvedilol displayed varying degrees of inverse agonism on the Gly389 variant of the receptor (i.e., actively switching off the β₁-AR), surprisingly, only carvedilol showed very specific and marked inverse agonist effects on the more frequent Arg389 variant. These specific effects of carvedilol on the Arg389 variant of the β₁-AR were also seen for control of beating frequency in rat cardiac myocytes expressing the 2 receptor variants. This FRET sensor permitted direct observation of activation of the β₁-AR in living cells in real time. It revealed that β₁-AR variants dramatically differ in their responses to diverse beta blockers, with possible consequences for their clinical use.