Calmodulin is a ubiquitous Ca2+ binding protein that binds to ryanodine rectors (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 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 calmodulin did not alter the time dependence of Ca2+ spark appearance. These results indicate that calmodulin may not be essential for RyR1-dependent Ca2+ release in adult mammalian skeletal muscle.