Five different subunits, alpha, beta, gamma, delta and epsilon, constitute the acetylcholine receptors from mammalian skeletal muscle. Their corresponding mRNA levels are regulated differentially. In particular, mRNAs encoding the gamma- and epsilon-subunits, which specify two AChR isoforms, show a reciprocal behaviour during synapse formation and maturation. We have isolated 5' flanking sequences of the gamma- and epsilon-subunit genes that confer muscle-specific expression upon transient transfection of primary cultures of rat muscle cells. The gamma-subunit gene fragment contains two adjacent CANNTG sequence motifs that are essential for muscle-specific transcriptional activity suggesting transactivation by helix-loop-helix proteins. The epsilon-subunit gene fragment carries only a single CANNTG consensus motif which is not required for expression in transfected muscle cells. This sequence motif is, however, necessary to repress transcriptional activity in non-muscle cells and thus may control the muscle-specific expression of the epsilon-subunit gene. The results suggest that CANNTG motifs together with their 3' and 5' flanking nucleotides provide binding sites for both activating as well as repressing trans-acting factors. These elements could thus contribute to the muscle-specific expression of AChR subunit genes.