The neuropeptide calcitonin gene-related peptide calcitonin gene-related peptide, and muscle electrical activity regulate in opposite directions the content of nicotinic acetylcholine receptor α-subunit mRNA in primary cultures of chick embryonic myotubes. Indeed, treating the cells with calcitonin generelated peptide or blocking the spontaneous activity of muscle cells by tetrodotoxin (an inhibitor of sodium channels) increases, although to different levels, the content of acetylcholine receptor α-subunit mRNA [Fontaine B., Klarsfeld A. and Changeux J. P. (1987) J. Cell Biol. 105, 1337–1342; Klarsfeld A. and Changeux J. P. (1985) Proc. natn. Acad. Sci. U.S.A. 82, 4558–4562]. In this paper, we demonstrate that, under these in vitro culture conditions, calcitonin gene-related peptide (0.1 μM) and tetrodotoxin (0.5 μM) regulate to a smaller extent (no more than 2.5-fold above control) the levels of acetylcholine receptor γ- and δ-subunit mRNAs. No effect of either compound on acetylcholine receptor biosynthesis was observed during the initial three days of culture. The response to calcitonin gene-related peptide was already maximal when the cells were treated between days three and four after plating (about 3-fold increase of the α-subunit mRNA level). The effect of tetrodotoxin resulted in a six-fold increase of the acetylcholine receptor α-subunit mRNA level in cells treated between days three and four, and still increased when the cells were exposed to tetrodotoxin through days six and eight (up to a maximum of 20-fold). Interestingly, the development of the response to tetrodotoxin coincided with a marked decline of the basal level of α-subunit mRNA, which, most likely, corresponded to the onset of spontaneous electrical activity in these cultures. Results of RNase protection experiments suggest that both calcitonin gene-related peptide and electrical activity regulate α-subunit biosynthesis, at least in part, at the transcriptional level. These results are discussed in the context of the mechanisms which control expression of the genes coding for acetylcholine receptor subunits during muscle differentiation and motor endplate development.