The potential for luminal Ca2+ to modulate the activity of both SERCA1 pumps and RyR1 channels was examined in isolated skeletal muscle heavy sarcoplasmic reticulum vesicles. A synchronous assay was developed using Calcium Green-2 to monitor extravesicular Ca2+ and NADH oxidation as an indicator of SERCA1 activity. Results from these assays demonstrated that SERCA1 pumps responded with near-maximal activity to RyR1-mediated Ca 2+ leak states. Therefore the activities of both the SERCA1 and the RyR1 appear intertwined through a luminal, Ca2+ mediated, cross talk, such that the formation of SERCA1-mediated luminal Ca2+ gradients required for RyR1-mediated Ca2+ releases is dependent upon the distribution of Ca2+ between free and bound luminal Ca2+ compartments. (Abstract shortened by UMI.)