Enhancement of presynaptic Ca²⁺ signals is widely recognized as a potential mechanism for heterosynaptic potentiation of neurotransmitter release. Here, we show that stimulation of a serotonergic interneuron increased spike-evoked Ca²⁺ in a manner consistent with its neuromodulatory effect on synaptic transmission. In the gastropod mollusc, Tritonia diomedea, stimulation of a serotonergic dorsal swim interneuron (DSI) at physiological rates heterosynaptically enhances the strength of output synapses made by another swim interneuron, C2, onto neurons in the pedal ganglion. Using intracellular electrophysiological recording combined with real-time confocal imaging of C2 (loaded with Oregon Green Bapta 1), it was determined that DSI stimulation increases the amplitude of spike-evoked Ca2+ signals in C2 without altering basal Ca²⁺ signals. This neuromodulatory action was restricted to distal neurites of C2, where synapses with pedal neurons are located. The effect of DSI stimulation on C2 spike-evoked Ca²⁺ signals resembled DSI heterosynaptic enhancement of C2 synapses in several measures: both decayed within 15 seconds, both were abolished by the serotonin receptor antagonist, methysergide, and both were independent of DSI's depolarizing actions on C2. A brief puff of serotonin could mimic the enhancement of spike-evoked Ca²⁺ signals in the distal neurites of C2, but larger puffs or bath-applied serotonin elicited non-physiological effects. These results suggest that DSI heterosynaptic enhancement of C2 synaptic strength may be mediated by a local enhancement of spike-evoked Ca²⁺ signals in the distal neurites of C2.