Calmodulin is a ubiquitous Ca²⁺ binding protein that binds to RyR and is thought to modulate its activity. Here we evaluated the effects of recombinant calmodulin on the rate of occurrence and spatial properties of Ca²⁺ sparks as an assay of sarcoplasmic reticulum Ca²⁺ release channel, ryanodine receptor, activation in saponin permeabilized mouse myofibers. Control myofibers exhibited a time dependent increase and subsequent decrease in spark frequency. Recombinant wild-type calmodulin prevented the time dependent appearance of Ca²⁺ sparks and decreased the derived Ca²⁺ flux from the sarcoplasmic reticulum during a spark by approximately 37%. A recombinant Ca²⁺ insensitive form of calmodulin resulted in an instantaneous increase in spark frequency as well as an increase in the derived Ca²⁺ flux by approximately 24%. Endogenous calmodulin was found to primarily localize to the Z-line. Surprisingly, removal of endogenous CaM did not alter the time dependence of Ca²⁺ spark appearance. These results indicate that CaM may not be essential for RyR1 dependent Ca²⁺ release in adult mammalian skeletal muscle.