Brief elevations of myoplasmic Ca2+ (Ca2+ sparks) have been visualized in embryonic mammalian skeletal muscle but are rarely seen in adult skeletal muscle (Conklin et al., 1998; Gyorke and Gyorke, 1996; Shirokova et al. 1998). I have monitored the occurrence of Ca2+ sparks in dissociated mouse diaphragm and the hindlimb (EDL) muscle fibers as a function of development using a confocal microscope and the fluorescent dye, Fluo4-AM. Ca2+ sparks were identified from many series of XY images of fiber sections using a computer analysis program. The frequency of events decreased to significantly low levels within two weeks in the diaphragm fibers and within 1 week in EDL fibers. The spatial properties of events in XY images demonstrated a decreasing trend as development progressed, even though there were no significant differences in temporal parameters of events measured in line-scan mode. To demonstrate that the occurrence of Ca2+ sparks in embryonic skeletal muscle was not related to the isoform type of RyR, Western blot analysis was performed on diaphragm muscles at developmental ages corresponding to Ca2+ spark frequency data. The decline of Ca 2+ spark frequency did not correlate with RyR3 expression suggesting that the event frequency is not attributed to RyR isoform. To further elucidate the production of Ca2+ sparks in embryonic mammalian skeletal muscle, embryonic diaphragm fibers (E18) were bathed in Ringer's solution containing 0Ca2+ with 1 mM EGTA, 1.8mM Ca2+ (physiological Ca2+ concentration) or 8mM Ca2+. The Ca2+ event frequency increased in fibers bathed in solution containing high concentrations of external Ca2+ and decreased in fibers bathed in 0Ca2+ indicating that Ca2+ influx could modulate the frequency of Ca2+ events in embryonic mammalian skeletal muscle. To confirm this finding, Ca2+ channels were blocked using CoCl2 and specifically the L-type Ca 2+ channels were inhibited using the dihydropyridine antagonist, nifedipine. The frequency of Ca2+ events decreased significantly after treatment with both types of Ca2+ channel blockers, indicating that Ca2+ spark production in embryonic mouse skeletal muscle is influenced by Ca2+ influx from L-type Ca2+ channels.