The modulation of the prefrontal cortex by the neurotransmitter serotonin (5-HT) is thought to play a key role in determining adult anxiety levels. Layer II/III of the prefrontal cortex, which mediates communication across cortical regions, displays a high level of 5-HT(1A) receptor binding in normal individuals and a significantly lower level in patients with mood and anxiety disorders. Here, we examine how serotonin modulates pyramidal neurons in layer II/III of the rat prefrontal cortex throughout postnatal development and in adulthood. Using whole cell recordings in brain slices of the rat medial prefrontal cortex, we observed that serotonin directly inhibits layer II/III pyramidal neurons through 5-HT(1A) receptors across postnatal development (postnatal days 6-96). In adulthood, a sex difference in these currents emerges, consistent with human imaging studies of 5-HT(1A) receptor binding. We examined the effects of early life stress on the 5-HT(1A) receptor currents in layer II/III. Surprisingly, animals subjected to early life stress displayed significantly larger 5-HT(1A)-mediated outward currents throughout the third and fourth postnatal weeks after elevated 5-HT(1A) expression during the second postnatal week. Subsequent exposure to social isolation in adulthood resulted in the almost-complete elimination of 5-HT(1A) currents in layer II/III neurons suggesting an interaction between early life events and adult experiences. These data represent the first examination of functional 5-HT(1A) receptors in layer II/III of the prefrontal cortex during normal development as well as after stress.