The effect of serotonin (5-HT) on the release of glutamate was examined in pyramidal cells in layers II-VI of the frontal cortex. The intracellular recording electrode contained 1% biocytin so the neurons could later be visualized with an avidin-biotin peroxidase method. Pyramidal cells in layer V of the frontal cortex showed the greatest 5-HT-induced increase in both the frequency and amplitude of 'spontaneous' (non-electrically evoked) excitatory post-synaptic currents (EPSCs). A small proportion of neurons in layer II/III showed an increase in EPSC frequency, whereas cells in layer VI showed no significant change in either EPSC frequency or amplitude. The physiological response to 5-HT mirrors the high density of 5-HT(2A) receptors in layer V, as well as the pattern of thalamic projections in frontal cortex. The specific induction of EPSCs in layer V neurons suggests that 5-HT preferentially modulates the output neurons of the frontal cortex.