Relationships between nicotinic acetylcholine receptor (nAChR) channel function and nAChR subunit mRNA expression were explored in acutely isolated rat medial habenula (MHb) neurons using a combination of whole-cell recording and single cell RT-PCR techniques. Following amplification using subunit-specific primers, subunits could be categorized in one of three ways: (i) present in 95-100% cells: α3, α4, α5, β2 and β4; (ii) never present: α2; and (iii) sometimes present (~40% cells): α6, α7 and β3. These data imply that α2 subunits do not participate in nAChRs on MHb cells, that α6, α7 and β3 subunits are not necessary for functional channels but may contribute in some cells, and that nAChRs may require combinations of all or subsets of α3, α4, α5, β2 and β4 subunits. Little difference in the patterns of subunit expression between nicotine-sensitive and insensitive cells were revealed based on this qualitative analysis, implying that gene transcription per se may be an insufficient determinant of nAChR channel function. Normalization of nAChR subunit levels to the amount of actin mRNA, however, revealed that cells with functional channels were associated with high levels (>0.78 relative to actin; 11/12 cells) of all of the category (i) subunits: α3, α4, α5, β2 and β4. Conversely, one or more of these subunits was always low (<0.40 relative to actin) in all cells with no detectable response to nicotine. Thus the formation of functional nAChR channels on MHb cells may require critical levels of several subunit mRNAs. Copyright (C) 2000 Elsevier Science Ltd.