As a neurotransmitter and neuromodulator, serotonin (5-HT) influences neuronal outgrowth in the nervous systems of several species. In PC12 cells, 5-HT is known to have neuritogenic effects, although the signal transduction pathway responsible for these effects is not understood. In this study, we hypothesized that a 5-HT-induced increase in intracellular Ca2+ concentration ([Ca2+]i) could be involved in mediating the effects of 5-HT. Application of 5-HT to PC12 cells enhanced nerve growth factor (NGF)-induced neurite outgrowth in a dose-dependent manner, and the sensitivity of this neuritogenic effect was increased in differentiated PC12 cells. In accordance, an increase in [Ca2+]i was observed following application of 5-HT in differentiated PC12 cells. This increase was amplified by further NGF treatment. 5-HT-induced increases in [Ca2+]i were inhibited by MDL 72222, a selective 5-HT3 receptor antagonist, and nifedipine, an L-type calcium channel blocker, but not by ketanserin, a 5-HT2 receptor antagonist, or thapsigargin, a specific inhibitor of endoplasmic reticulum Ca2+-ATPase. These pharmacological tests indicated that 5-HT-induced increases in [Ca2+]i are mediated by activation of voltage-gated calcium channels via 5-HT3 receptors and that 5-HT-induced increases in [Ca2+]i are likely to be independent of activation of 5-HT2 receptors in PC12 cells. Furthermore, the neuritogenic effect of 5-HT was suppressed by MDL 72222, nifedipine, calmodulin (CaM) inhibitor, and calcineurin inhibitors. Taken together, our results indicate that 5-HT-induced increases in [Ca2+]i, which are mediated via 5-HT3 receptors and L-type calcium channels in PC12 cells, and subsequent activation of CaM and calcineurin enhance NGF-induced neurite outgrowth.