The molecular makeup of the serotonin 5-HT₃ receptor (5-HT₃R) 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 4 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 4-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 4-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 4-nAChR subunits, we used the sulphydryl agent 2-aminoethyl methanethiosulphonate (MTSEA), which has previously been shown to modulate expressed 5-HT₃Rs that contain the 4-nAChR subunit. In half of the interneurons examined, MTSEA significantly enhanced the amplitude of the 5-HT₃R-mediated responses, which is consistent with the notion that the 4-nAChR subunit co-assembles with the 5-HT_3A subunit to form a native heteromeric 5-HT₃R channel in rat CA1 hippocampal interneurons in vivo. In addition, the single-channel properties of the 5-HT₃R 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.