Digitonin-permeabilized isolated neurohypophysial nerve terminals are known to release their secretory vesicle content under calcium challenge. On this preparation, we monitored intra-organelle Ca2+ concentration using digital fluorescence microscopy of Fura-2. The superfusion of artificial intracellular solution containing 10 to 50 microM Ca2+ induced an intra-organelle [Ca2+] increase. Two major organelles are candidates for this increase: secretory vesicles and mitochondria. In an attempt to detect calcium changes in the vesicles, ruthenium red was used to impair mitochondrial calcium uptake. Part of the ruthenium red-insensitive intra-organelle [Ca2+] increase was abolished by raising sodium in the solution. Removing sodium boosted the intra-organelle [Ca2+] increase. These results taken together suggest the participation of Na/Ca exchange, known to exist in the membrane of these secretory vesicles. In addition to Na/Ca exchange, there would be at least another mechanism of vesicular calcium intake, as suggested by the partial inhibition of intra-organelle [Ca2+] increase obtained under acidic compartments: neutralization with NH4Cl. This mechanism remains to be defined. The main conclusion presented here, that an intravesicular [Ca2+] increase takes place at the rate of secretion, was predicted by the hypothesis that intravesicular Ca2+ changes would be involved in stimulus-secretion coupling.