Controlled exposure to retinoic acid (RA) induces the murine embryonal carcinoma cell line P19S18O1A1 (P19) to differentiate into a variety of cell types. One of the cell types exhibits neuronal-like morphology and expresses neuronal markers including neurofilament proteins, glutamate receptors, and the cholinergic enzymes choline acetyl-transferase and acetylcholinesterase. In this study we use Northern blot analysis, double-label immunocytochemistry, and single cell RNA analysis using polymerase chain reaction to show that RA-treated P19 cells with neuronal-like morphology also express neuronal nicotinic acetylcholine receptor (nAChR) subunits alpha 3, alpha 4, and beta 2. Greater than 80% of RA-treated P19 cells with a neuronal-like phenotype express nAChR alpha 4 subunit transcripts and both alpha 4 and beta 2 protein. The RA-induced expression of alpha 3 transcripts accounts for a comparably small number of nAChR-containing cells (< 20%) of which half coexpress alpha 4 transcripts. Expression of high-levels of alpha 4 RNA is dependent upon both cell-cell contact and RA exposure. The appearance of nAChR subunits also coincides with RA-induced expression of high affinity [3H]-nicotine binding receptors. The P19 cell line offers an inducible neuronal cell system to study mammalian neuronal nicotinic receptor expression and the development of high affinity nicotinic binding sites similar to those expressed in the mammalian central nervous system.