Calmodulin (CaM) and S100A1 fine-tune skeletal muscle Ca release via opposite modulation of the ryanodine receptor type 1 (RyR1). Binding to and modulation of RyR1 by CaM and S100A1 occurs predominantly at the region ranging from amino acid residue 3614-3640 of RyR1 (here referred to as CaMBD2). Using synthetic peptides， it has been shown that CaM binds to two additional regions within the RyR1， specifically residues 1975-1999 and 4295-4325 (CaMBD1 and CaMBD3， respectively). Because S100A1 typically binds to similar motifs as CaM， we hypothesized that S100A1 could also bind to CaMBD1 and CaMBD3. Our goals were: (1) to establish whether S100A1 binds to synthetic peptides containing CaMBD1 and CaMBD3 using isothermal calorimetry (ITC)， and (2) to identify whether S100A1 and CaM modulate RyR1 Ca release activation via sites other than CaMBD2 in RyR1 in its native cellular context. We developed the mouse model (RyR1D-S100A1KO)， which expresses point mutation RyR1-L3625D (RyR1D) that disrupts the modulation of RyR1 by CaM and S100A1 at CaMBD2 and also lacks S100A1 (S100A1KO). ITC assays revealed that S100A1 binds with different affinities to CaMBD1 and CaMBD3. Using high-speed Ca imaging and a model for Ca binding and transport， we show that the RyR1D-S100A1KO muscle fibers exhibit a modest but significant increase in myoplasmic Ca transients and enhanced Ca release flux following field stimulation when compared to fibers from RyR1D mice， which were used as controls to eliminate any effect of binding at CaMBD2， but with preserved S100A1 expression. Our results suggest that S100A1， similar to CaM， binds to CaMBD1 and CaMBD3 within the RyR1， but that CaMBD2 appears to be the primary site of RyR1 regulation by CaM and S100A1.