The effects of serotonin (5-HT) on neuronal activity were examined during postnatal development in layer V pyramidal neurons of the rat prefrontal cortex (PFC) in vitro. Whole-cell patch-clamp recordings were made in slices obtained from rats aged between postnatal day (P) 6 and P31. In P14 or younger neurons, bath application of 5-HT (10 μm) induced a large depolarization followed by tonic firing at 2–5 Hz. The excitatory effects of 5-HT decreased rapidly after P14, so that by P21, 5-HT produced a small depolarization or hyperpolarization without cell firing. The excitatory effects of 5-HT at younger ages were attributed to 5-HT_2A receptors because the effects were mimicked by the 5-HT₂ agonist α-methyl-5-HT but not by the 5-HT₃ agonist 1-(m-chlorophenyl)-biguanide, nor by the 5-HT_2B/2C agonist 1-(3-chlorophenyl)piperazine, and were blocked by the 5-HT_2A antagonists ketanserin and α-phenyl-1-(2-phenylethyl)-4-piperidinemethanol. The excitatory responses persisted in 0 [Ca²⁺]_o and high [Mg²⁺]_o in the presence of TTX or blockers of ionotropic glutamate receptors, suggesting that the effects were mediated essentially by postsynaptic mechanisms. The responses to 5-HT involve a reduction of K⁺ conductance and an enhancement of the hyperpolarization-activated Na⁺/K⁺ current. The developmental decline of 5-HT-induced excitatory effects was associated with a downregulation of 5-HT_2A receptor function and a decrease in the input resistance during early life. These results suggest that 5-HT is an important regulator of neuronal activity in the neonatal PFC and may play a role in activity-dependent developmental processes.