The modulation by internal free [Mg2+] of spontaneous calcium release events (Ca2+ "sparks") from the sarcoplasmic reticulum (SR) was studied in depolarized notched frog skeletal muscle fibers using a laser scanning confocal microscope in line-scan mode (x vs. t). Over the range of [Mg2+] from 0.13 to 1.86 mM, decreasing the [Mg2+] induced an increase in the frequency of calcium release events in proportion to [Mg2+]-1.6. The change of event frequency was not due to changes in [Mg-ATP] or [ATP]. Analysis of individual SR calcium release event properties showed that the variation in event frequency induced by the change of [Mg2+] was not accompanied by any changes in the spatiotemporal spread (i.e., spatial half width or temporal half duration) of Ca2+ sparks. The increase in event frequency also had no effect on the distribution of event amplitudes. Finally, the rise time of calcium sparks was independent of the [Mg2+], indicating that the open time of the SR channel or channels underlying spontaneous calcium release events was not altered by [Mg2+] over the range tested. These results suggest that in resting skeletal fibers, [Mg2+] modulates the SR calcium release channel opening frequency by modifying the average closed time of the channel without altering the open time. A kinetic reaction scheme consistent with our results and those of bilayer and SR vesicle experiments indicates that physiological levels of resting Mg2+ may inhibit channel opening by occupying the site for calcium activation of the SR calcium release channel.