Enhancement of contractile force (inotropy) occurs in skeletal muscle following neuro-endocrine release of catecholamines and activation of muscle b-adrenergic receptors. Despite extensive study over decades, the molecular mechanism underlying the inotropic response in skeletal muscle is not understood. We tested the hypothesis that, following acute b-receptor stimulation, cAMP-dependent protein kinase A (PKA) phosphorylation of the sarcoplasmic reticulum (SR) Ca²⁺ release channel/ryanodine receptor type 1 (RyR1) is essential for muscle inotropy. For this purpose, we generated RyR1-S2844A mice in which the Serine at the PKA phosphorylation site (S2844) in the RyR1 was replaced with an Alanine that cannot be phosphorylated. Contractile force was measured in isolated Extensor Digitorum Longus (EDL) muscles and myoplasmic Ca²⁺ was measured in single Flexor Digitorum Brevis (FDB) muscles that was loaded with the cell permeable fluorescent Ca²⁺ indicator Fluo-4AM. Treating skeletal muscle from wild-type mice with the b-receptor agonist isoproterenol (ISO; 1mM) increased RyR1 PKA phosphorylation, myoplasmic twitch Ca²⁺ (1194% of the pre- exposure control; n=8, P<0.01) and contractile force (control, 1407.8 kPa; ISO, 1597.6 kPa; mean  SEM, n=8, P<0.01). In contrast, RyR1-S2844A mice displayed abrogated responses to ISO on twitch Ca²⁺ (ISO, 1034% of the pre exposure control; n=10 cells from 3 animals, P=n.s.) and force (RyR1-S2844A control, 14518 kPa; RyR1-S2844A ISO, 14216 kPa; mean  SEM, n=8, P=n.s.). In vivo muscle strength was lower in RyR1-S2844A mice compared to WT following stress. These data suggest that the molecular mechanism underlying skeletal muscle inotropy requires enhanced SR Ca²⁺ release due to PKA phosphorylation of serine 2844 in RyR1.