Second messenger regulation of neuronal acetylcholine receptors (AChRs) was investigated in a mouse fibroblast cell line, M10, stably transfected with chicken alpha 4 and beta 2 cDNAs. Both forskolin and 8-bromo-cyclic adenosine 3',5'-monophosphate (cAMP) induced large increases in the numbers of AChRs. The increases were due in part to increased transcription and translation of the alpha 4 and beta 2 genes. Blockade of protein synthesis with cycloheximide, however, revealed that forskolin also exerts a post-translational effect, increasing the number of surface receptors by twofold. Immunoblot analysis of sucrose gradient fractions confirmed that the cells had a large fraction of unassembled subunits potentially available for receptor assembly. The post-translational effect of forskolin was blocked by H-89, an inhibitor of cAMP-dependent protein kinase, and by okadaic acid, an inhibitor of phosphatases 1 and 2A. Nicotine also acted post-translationally to induce a twofold increase in the number of surface receptors, but the mechanism differed from that utilized by forskolin, since the effects of the two agents were additive and were differentially affected by okadaic acid. The results suggest that protein phosphorylation-dephosphorylation mechanisms act post-translationally to increase the number of neuronal AChRs maintained on the cell surface. This could be achieved by increasing the efficiency of receptor assembly, transport, or stabilization on the cell surface.