Serotonin (5-HT) production and expression of 5-HT receptors (5-HTRs) occur early during prenatal development. Recent evidence suggests that, in addition to its classical role as a neurotransmitter, 5-HT regulates neuronal connectivity during mammalian development by modulating cell migration and neuronal cytoarchitecture. Given the variety of 5-HTRs, researchers have had difficulty clarifying the specific role of each receptor subtype in brain development. Signalling mediated by the G-protein-coupled 5-HT1AR and 5-HT7R, however, has been associated with neuronal plasticity. Thus, we hypothesized that 5-HT promotes neurite outgrowth through 5-HT1AR and 5-HT7R. The involvement of 5-HT1AR and 5-HT7R in the morphology of rat hippocampal neurons was evaluated by treating primary cultures at 2 days in vitro with 5-HT and specific antagonists for 5-HT1AR and 5-HT7R (WAY-100635 and SB269970, respectively). The stimulation of hippocampal neurons with 100 nM 5-HT for 24 hr produced no effect on either the number or the length of primary neurites. Nonetheless, after 5HT7R was blocked, the addition of 5-HT increased the number of primary neurites, suggesting that 5HT7R could inhibit neuritogenesis. In contrast, 5-HT induced secondary neurite outgrowth, an effect inhibited by 1 μM WAY-100635 or SB269970. These results suggest that both serotonergic receptors participate in secondary neurite outgrowth. We conclude that 5-HT1AR and 5-HT7R regulate neuronal morphology in primary hippocampal cultures by promoting secondary neurite outgrowth. © 2014 Wiley Periodicals, Inc.