The three principal sarcoplasmic/endoplasmic reticulum Ca++ pump inhibitors have been compared for their effects on Ca++ fluxes across intracellular stores present in isolated skeletal muscle and brain membrane preparations. At moderate concentrations that only partially inhibited Ca++ pumping, all three inhibitors induced transient release of Ca++ from isolated sarcoplasmic reticulum membranes, and release was ruthenium red-sensitive, much faster and sustained at higher pump inhibitor concentrations. In contrast, in unidirectional 45Ca efflux assays, cyclopiazonic acid appeared to have little effect, thapsigargin decreased efflux and 2,5-di(tert-butyl)-1,4-benzohydroquinone increased efflux only slightly. These observations taken together suggest that transient releases were manifest primarily by vesicles with a high ratio of ryanodine receptors to pumps (and thus more susceptible to becoming leaky with only some pumps inhibited), and that Ca(++)-induced Ca++ release amplified releases when all pumps were blocked. These mostly indirect side effects were specific for ryanodine receptors. In similar experiments with brain cerebellar membranes, none of the three inhibitors appeared to directly reduce release induced by inositol 1,4,5-trisphosphate. These findings may affect interpretation of results of experiments involving application of these compounds to isolated membranes, cells or tissue preparations.