The serotonin 5-HT3 receptor, a ligand-gated ion channel, has previously been shown to be present on a subpopulation of brain nerve terminals, where, on activation, the 5-HT3 receptors induce Ca2+ influx. Whereas postsynaptic 5-HT3 receptors induce depolarization, being permeant to Na+ and K+, the basis of presynaptic 5-HT3 receptor-induced calcium influx is unknown. Because the small size of isolated brain nerve terminals (synaptosomes) precludes electrophysiological measurements, confocal microscopic imaging has been used to detect calcium influx into them. Application of 100 nM 1-(m-chlorophenyl)biguanide (mCPBG), a highly specific 5-HT3 receptor agonist, induced increases in internal free Ca2+ concentration ([Ca2+]i) and exocytosis in a subset of corpus striatal synaptosomes. mCPBG-induced increases in [Ca2+]i ranged from 1.3 to 1.6 times over basal values and were inhibited by 10 nM tropisetron, a potent and highly specific 5-HT3 receptor antagonist, but were insensitive to the removal of external free Na+ (substituted with N-methyl-D-glucamine), to prior depolarization induced on addition of 20 mM K+, or to voltage-gated Ca2+ channel blockade by 10 microM Co2+/Cd2+ or by 1 microM omega-conotoxin MVIIC/1 microM oemga-conotoxin GVIA/200 nM agatoxin TK. In contrast, the Ca2+ influx induced by 5-HT3 receptor activation in NG108-15 cells by 1 microM mCPBG was substantially reduced by 10 microM Co2+/Cd2+ and was completely blocked by 1 microM nitrendipine, an L-type Ca2+ channel blocker. We conclude that in contrast to the perikaryal 5-HT3 receptors, presynaptic 5-HT3 receptors appear to be uniquely calcium-permeant.