Previous studies have outlined an important role for serotonin (5-HT) in the development of synaptic connectivity and function in the cerebral cortex. In this study, we have examined the effects of 5-HT on synaptic function in prefrontal cortex at a time of intense synapse formation and remodeling. Whole-cell recordings in slices derived from P16-P20 animals showed that administration of 5-HT induced a robust increase in synaptic activity that was blocked by CNQX but not by bicuculline. This 5-HT-induced increase in glutamate-mediated synaptic activity was pharmacologically heterogeneous as it was differentially inhibited by the receptor subtype selective antagonists SB-269970, MDL 100907 and GR 113808 and thus involved 5-HT7, 5-HT2A and 5-HT4 receptors. These results, obtained in juvenile cortex, contrast with those seen in adults where the increase in sEPSCs was mediated solely by 5-HT2A receptors. In developing cortex, activation of 5-HT7, but not 5-HT2A or 5-HT4 receptors, elicited a robust inward current. However, the facilitation of synaptic activity mediated by all three of these receptors involved increases in both the amplitude and frequency of sEPSC and was blocked by TTX. These results are best interpreted as indicating that all three receptor subtypes increase synaptic activity by exciting neuronal elements within the slice. No evidence was found for a postsynaptic facilitation of synaptic currents by 5-HT. Together, these results show that the repertoire of electrophysiologically-active 5-HT receptors in prefrontal cortex is developmentally regulated, and that 5-HT7 and 5-HT4 receptors play a previously unsuspected role regulating synaptic activity in this region.