Fura-2 microfluorometric measurements of calcium concentrations in excitor motor nerve terminals of the first walking leg of crayfish Procambrus clarkii were made during application of serotonin (5-HT) to examine whether changes in residual calcium concentration or calcium influx are responsible for the 5-HT-induced increase in transmitter release at this synapse. Calcium concentrations and EPSPs were monitored simultaneously during 0.5-Hz and moderate-frequency (3–16 Hz) stimulation before, during, and after a 5-min application of 5-HT at concentrations between 5 and 100 microM. We did not observe a significant increase in resting calcium concentration [Ca2+]i in presynaptic terminals during or after application of 5-HT despite simultaneously recorded excitatory junction potential (EJP) enhancement. Injection of the calcium buffer EGTA to concentrations sufficient to reduce significantly the buildup of frequency-dependent facilitation during trains of action potentials did not reduce 5-HT- mediated EJP enhancement. We therefore conclude that 5-HT does not release calcium from intracellular stores to an extent sufficient to overcome calcium buffering and removal systems. Spontaneous release of transmitter quanta was increased by 5-HT to the same extent in normal and increased-osmolarity saline in the absence of measured changes in [Ca2+]i. Thus, changes in residual calcium cannot explain 5-HT-induced synaptic enhancement. Increasing the action-potential-mediated calcium influx by broadening the spike or by increasing extracellular calcium increased evoked transmitter release and increased the accumulation of calcium during trains of action potentials. However, though 5-HT produced a comparable synaptic enhancement, we found no evidence for increased calcium accumulation during trains of action potentials, suggesting that net calcium influx, buffering, and removal are not affected by 5-HT. Therefore, we conclude that 5-HT's action does not result from effects on calcium metabolism and suggest that it acts on the neurosecretory apparatus to increase the effectiveness of calcium for stimulating release. Furthermore, based on its relatively constant effects on facilitated and unfacilitated action-potential-evoked release and spontaneous transmitter release, we conclude that 5-HT actions are independent of the concentration of calcium that is driving release.