Sperm-egg fusion induces an intracellular free calcium concentration ([Ca2+]i) increase and exocytosis of cortical granules (CGs). Recently we used an impermeable fluorescent membrane probe, 1-[4-(trimethylammonio)phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH), to develop a method to evaluate the kinetics of exocytosis in single living cells. In this study we used digital imaging and confocal laser scanning microscopy to evaluate CG exocytosis in living mouse eggs with TMA-DPH. Time-related changes of CG exocytosis were estimated as the percent increase of TMA-DPH fluorescence. The increase of fluorescence in the egg started after sperm attachment, continued at an almost uniform rate, and ceased at 45-60 min. Whereas the [Ca2+]i increase at fertilization was transient or oscillatory, exocytosis was not always induced concomitantly with each [Ca2+]i peak. Next we used this method to determine some intracellular mediators of exocytosis in the egg. An intracellular calcium chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester, and a microfilament inhibitor, cytochalasin B, blocked sperm-induced exocytosis. A guanosine 5'-triphosphate-binding protein activator, AlF4-, induced exocytosis. These results suggest that [Ca2+]i, microfilament, and guanosine 5'-triphosphate-binding proteins may be involved in CG exocytosis. In conclusion, this method has significant advantages for studying exocytosis in living eggs.