To further elucidate the role of the neuromodulatory transmitter serotonin (5-HT) during early postnatal development of the neocortex, we investigated the effects of 5-HT on gap junction coupling in the somatosensory cortex of rats aged between postnatal days 7 and 10. The gap junction-permeable tracer neurobiotin was injected into single neurons via microelectrodes or patch pipettes. Under control conditions, clusters of about 25 tracer-coupled neurons were observed. Serotonin reduced dye-coupling between lamina II/III pyramidal cells in a concentration-dependent and reversible manner. The 1,4,5-inositol triphosphate (IP3) receptor antagonist heparin as well as the protein kinase C inhibitor NPC 15437 suppressed the uncoupling action of 5-HT, suggesting that the serotonergic effect involved IP3 receptor-mediated release of calcium ions from intracellular stores. In contrast, the 5-HT-induced reduction in gap junction coupling was not antagonized by Rp-adenosine3′,5′-cyclic monophosphothionate, an inhibitor of CAMP dependent protein kinase. The uncoupling effect of 5-HT was mimicked by 5-HT2 receptor agonists and antagonized by the 5-HT2 receptor antagonist ritanserin, indicating that 5-HT suppressed gap junction coupling via activation of 5-HT2 class receptors. Our results suggest that the developmental functions of 5-HT not only involve the modulation of chemical synaptic transmission but also include the regulation of the gap junctional communication system during differentiation of the neocortex.