Enhancement of contractile force (inotropy) occurs in skeletal muscle following neuroendocrine release of catecholamines and activation of muscle β-adrenergic receptors. Despite extensive study, the molecular mechanism underlying the inotropic response in skeletal muscle is not well understood. Here we show that phosphorylation of a single serine residue (S2844) in the sarcoplasmic reticulum (SR) Ca(2+) release channel/ryanodine receptor type 1 (RyR1) by protein kinase A (PKA) is critical for skeletal muscle inotropy. Treating fast twitch skeletal muscle from wild-type mice with the β-receptor agonist isoproterenol (isoprenaline) increased RyR1 PKA phosphorylation, twitch Ca(2+) and force generation. In contrast, the enhanced muscle Ca(2+), force and in vivo muscle strength responses following isoproterenol stimulation were abrogated in RyR1-S2844A mice in which the serine in the PKA site in RyR1 was replaced with alanine. These data suggest that the molecular mechanism underlying skeletal muscle inotropy requires enhanced SR Ca(2+) release due to PKA phosphorylation of S2844 in RyR1.