Intracellular recording from CA1 neurons of the rat hippocampal slice preparation was used to examine the possibility of functional interactions between 5-hydroxytryptamine (5-HT) and thyrotropin releasing hormone (TRH), which act as cotransmitters in other areas of the central nervous system. 5-HT (30 microM) elicited complex effects consisting of biphasic changes in membrane potential and a strong depression of the afterhyperpolarization (AHP) following a spike burst. TRH (10 microM) did not alter membrane potential or input conductance but it produced a partial block of the AHP. Under single-electrode voltage clamp, 5-HT and TRH both reduced the amplitude of voltage-activated total K+ currents. When the two substances were co-applied, their actions were occluded. The voltage-activated K+ current remaining in Ca(2+)-free solution lost its sensitivity to 5-HT and TRH, suggesting that the K+ current modulated by TRH and 5-HT was Ca(2+)-dependent, although TRH itself did not depress high-threshold voltage-activated Ca2+ currents. When a relatively small concentration (5 microM) of 5-HT was co-applied with an equimolar amount of TRH, the degree of block of the spike AHP was the sum of the two individual effects of these drugs. It is suggested that in hippocampal pyramidal cells 5-HT and TRH influenced neuronal excitability by depressing a Ca(2+)-dependent K+ current, a phenomenon perhaps mediated through a common intracellular second messenger pathway.