The molecular makeup of the serotonin 5-HT(3) receptor (5-HT(3)R) channel was investigated in rat hippocampal CA1 interneurons in slices using single-cell RT-PCR and patch-clamp recording techniques. We tested for the expression of the 5-HT(3A) (both short and long splice variants) and 5-HT(3B) subunits, as well as the expression of the alpha4 subunit of the neuronal nicotinic ACh receptors (nAChRs), the latter of which has been shown to co-assemble with the 5-HT(3A) subunit in heterologous expression systems. Both the 5-HT(3A)-short and alpha4-nAChR subunits were expressed in these interneurons, but we could not detect any expression of either the 5-HT(3B) or the 5-HT(3A)-long subunits. Furthermore, there was a strong tendency for the 5-HT(3A)-short and alpha4-nAChR subunits to be co-expressed in individual interneurons. To assess whether there was any functional evidence for co-assembly between the 5-HT(3A)-short and alpha4-nAChR subunits, we used the sulphydryl agent 2-aminoethyl methanethiosulphonate (MTSEA), which has previously been shown to modulate expressed 5-HT(3)Rs that contain the alpha4-nAChR subunit. In half of the interneurons examined, MTSEA significantly enhanced the amplitude of the 5-HT(3)R-mediated responses, which is consistent with the notion that the alpha4-nAChR subunit co-assembles with the 5-HT(3A) subunit to form a native heteromeric 5-HT(3)R channel in rat CA1 hippocampal interneurons in vivo. In addition, the single-channel properties of the 5-HT(3)R were investigated in outside-out patches. No resolvable single-channel currents were observed. Using non-stationary fluctuation analysis, we obtained an estimate of the single-channel conductance of 4 pS, which is well below that expected for channels containing both the 5-HT(3A) and 5-HT(3B) subunits.