In vitro, calmodulin (CaM) and S100A1 activate the skeletal muscle ryanodine receptor ion channel (RyR1) at submicromolar Ca2+concentrations, whereas at micromolar Ca2+concentrations, CaM inhibits RyR1. One amino acid substitution (RyR1-L3625D) has previously been demonstrated to impair CaM binding and regulation of RyR1. Here we show that the RyR1-L3625D substitution also abolishes S100A1 binding. To determine the physiological relevance of these findings, mutant mice were generated with the RyR1-L3625D substitution in exon 74, which encodes the CaM and S100A1 binding domain of RyR1. Homozygous mutant mice ( Ryr1D/D) were viable and appeared normal. However, single RyR1 channel recordings from Ryr1D/Dmice exhibited impaired activation by CaM and S100A1 and impaired CaCaM inhibition. Isolated flexor digitorum brevis muscle fibers from Ryr1D/Dmice had depressed Ca2+transients when stimulated by a single action potential. However, during repetitive stimulation, the mutant fibers demonstrated greater relative summation of the Ca2+transients. Consistently, in vivo stimulation of tibialis anterior muscles in Ryr1D/Dmice demonstrated reduced twitch force in response to a single action potential, but greater summation of force during high-frequency stimulation. During repetitive stimulation, Ryr1D/Dfibers exhibited slowed inactivation of sarcoplasmic reticulum Ca2+release flux, consistent with increased summation of the Ca2+transient and contractile force. Peak Ca2+release flux was suppressed at all voltages in voltage-clamped Ryr1D/Dfibers. The results suggest that the RyR1-L3625D mutation removes both an early activating effect of S100A1 and CaM and delayed suppressing effect of CaCaM on RyR1 Ca2+release, providing new insights into CaM and S100A1 regulation of skeletal muscle excitation-contraction coupling.