The effects of serotonin (5-HT) on neuronal responses to the excitatory amino acid agonist N-methyl-D-aspartate (NMDA) were examined in neocortical slices of the Fischer rat using current-clamp and single-electrode voltage-clamp techniques. Layer V neocortical neurons responded to application of NMDA by depolarization with no change or an apparent increase in input resistance. Following perfusion with 10(-5) M 5-HT, the response of these neurons to NMDA was significantly increased in both amplitude and duration, whereas neuronal responses to quisqualic acid and acetylcholine were not altered by 5-HT. Furthermore, the enhanced response to NMDA in 5-HT was long-lasting, and could not be reversed during the course of the experiment. Resting membrane potential and the postspike train afterhyperpolarization were not significantly altered by 5-HT, although the input resistance was decreased by 5-HT. Excitatory postsynaptic potentials (EPSPs) were usually not affected or reversibly decreased by 5-HT. However, in a few cells exhibiting a complex voltage-dependent EPSP, 5-HT produced a long-lasting enhancement in the amplitude of the EPSP. Under voltage-clamp conditions, with Na+- and K+-channels blocked, 5-HT enhanced the inward current stimulated by application of NMDA. It is suggested that 5-HT selectively enhances the voltage- and Ca2+-dependent NMDA response.