To study the effects of changes in sarcoplasmic reticulum (SR) intraluminal Ca2+ on the Ca2+ release mechanism, we correlated the activity of single cardiac ryanodine receptor (RyR) channels, monitored in planar bilayers, with the properties of spontaneous elementary Ca2+ release events (sparks) in intact ventricular myocytes, monitored by scanning confocal microfluorimetry. Under both normal conditions and Ca2+ overload, induced by elevation of extracellular [Ca2+], Ca2+ sparks represented single populations of events. During Ca2+ overload, the frequency of sparks increased from 0.8 to 3.1 events per second per 100 microm line scanned, and their amplitude increased from 100 nM to 400 nM. The duration of the Ca2+ sparks, however, was not altered. Changes in the properties of Ca2+ sparks were accompanied by only an approximately 30% increase in the SR Ca2+ content, as determined by emptying the intracellular Ca2+ stores using caffeine. When single Ca2+ release channels were incorporated into lipid bilayers and activated by cytoplasmic Ca2+ (approximately 100 nM) and ATP (3 mM), elevation of Ca2+ on the luminal side from 20 microM to 0.2-20 mM resulted in a 1.2-fold to 7-fold increase, respectively, in open probability (Po). This potentiation of Po was due to an increase in mean open time and frequency of events. The relative effect of luminal Ca2+ was greater at low levels of cytoplasmic [Ca2+] than at high levels of cytoplasmic [Ca2+], and no effect of luminal Ca2+ was observed to occur in channels activated by 0.5-50 microM cytoplasmic Ca2+ in the absence of ATP. Our results suggest that SR Ca2+ release channels are modulated by SR intraluminal Ca2+. These alterations in properties of release channels may account for, or contribute to, the mechanism of spontaneous Ca2+ release in cardiac myocytes