Confocal microscopy was used to assess internal calcium level changes in response to presynaptic receptor activation in individual, isolated nerve terminals (synaptosomes) from rat corpus striatum, focusing, in particular, on the serotonin 5-HT3 receptor, a ligand-gated ion channel. The 5-HT3 receptor agonist-induced calcium level changes in individual synaptosomes were compared with responses evoked by K+ depolarization. Using the fluorescent dye fluo-3 to measure relative changes in internal free Ca2+ concentration ([Ca2+]i), K+-induced depolarization resulted in variable but rapid increases in apparent [Ca2+]i among the individual terminals, with some synaptosomes displaying large transient [Ca2+]i peaks of varying size (two- to 12-fold over basal levels) followed by an apparent plateau phase, whereas others displayed only a rise to a sustained plateau level of [Ca2+]i (two- to 2.5-fold over basal levels). Agonist activation of 5-HT3 receptors induced slow increases in [Ca2+]i (rise time, 15-20 s) in a subset (approximately 5%) of corpus striatal synaptosomes, with the increases (averaging 2.2-fold over basal) being dependent on Ca2+ entry and inhibited by millimolar external Mg2+. We conclude that significant increases in brain nerve terminal Ca2+, rivaling that found in response to excitation by depolarization but having distinct kinetic properties, can therefore result from the activation of presynaptic ligand-gated ion channels.