The effect of various Ca(2+)-channel blockers on exocytosis has been studied at the level of single presynaptic terminals in rat hippocampal cell cultures. The fluorescence change of the styryl dye FM 1-43 has been used as a measure of exocytosis during electrical stimulation. omega-Conotoxin GVIA (2-10 microM) completely inhibited exocytosis in approximately 45% of the boutons in the field of view, while in approximately 55% exocytosis was inhibited incompletely (by 38%). This heterogeneity in response of presynaptic boutons was not seen with isradipine (5 microM) or omega-agatoxin IVA (80 nM), which inhibited exocytosis by 23% and 17%, respectively. However, it was observed with a combination of all three blockers. Pre- and postsynaptic events could be separated in single synapses by measuring FM1-43 release and NMDA-induced changes in the intracellular Ca2+ concentration independently.