Forskolin treatment of cells expressing Torpedo acetylcholine receptors leads to enhanced assembly efficiency of subunits, which correlates with increased phosphorylation of the gamma subunit. To determine the role of the two potential protein kinase A sites of the gamma subunit in receptor assembly, cell lines expressing different mutant receptors were established. Mouse fibroblast cell lines stably expressing wild-type Torpedo acetylcholine receptor alpha, beta, delta subunits plus one of three gamma subunit mutations (S353A, S354A, or S353,354A) were established to identify the protein kinase A phosphorylation sites of gamma in vivo, and to determine if increased phosphorylation of the gamma subunit leads to enhanced expression of receptors. We found that both serines (353, 354) in gamma are phosphorylated in vivo by protein kinase A, however, phosphorylation of either or both of these sites does not lead to increased assembly efficiency. We established a cell line expressing alpha, beta, and gamma(S353,354A) subunits only (no delta), and found that the presence of delta (or its phosphorylation) is also not necessary for the observed stimulation by forskolin. alpha beta gamma, alpha gamma, and beta gamma associations were stimulated by forskolin but alpha beta and alpha delta interactions were not. These data imply that the presence of gamma is necessary for forskolin action. We postulate that forskolin may stimulate acetylcholine receptor expression through a cellular protein that is involved in the folding and/or assembly of protein complexes, and that forskolin may regulate the action of such a protein through phosphorylation.