In spite of a growing understanding of the actions of 5-hydroxytryptamine (5-HT) in the prefrontal cortex, the specific cellular mechanism used by 5-HT in this region remains poorly understood. Previous studies have shown that 5-HT inhibits the afterhyperpolarization that follows a burst of spikes in pyramidal neurons. In the present study, we have used whole cell recordings in rat and mouse brain slices to re-examine this phenomenon with special emphasis on identifying the 5-HT receptor subtypes mediating this effect. Layer V pyramidal neurons display complex afterhyperpolarizations that are mediated predominantly by calcium-activated potassium channels and involve two distinct currents known as medium afterhyperpolarizating current and slow afterhyperpolarizating current (IsAHP). Administration of 5-HT reduced the current underlying these afterhyperpolarizations by selectively inhibiting IsAHP. Pharmacological analysis of this response indicates that the main receptor responsible for this inhibition belongs to the 5-HT2A subtype. Thus, α-methyl-5-HT and 2,5-dimethoxy-4-bromoamphetamine (DOB) mimic the effect of 5-HT and the effect of these agonists is blocked by MDL 100 907. Similarly, administration of α-methyl-5-HT is without effect in slices derived from 5-HT2A receptor knockout mice. However, 5-HT2A receptor blockade only partially suppressed the ability of 5-HT to inhibit IsAHP. This suggests the involvement of at least one more receptor subtype in this response. Consistent with this idea, administration of 5-carboxyamido-tryptamine, an agonist exhibiting no detectable affinity for 5-HT2A receptors, was also capable of suppressing IsAHP. These results identify 5-HT2A receptors as being primarily involved in mediating the 5-HT-induced inhibition of IsAHP in prefrontal cortex, while also recognizing a contribution by an additional 5-HT receptor subtype.