AbstractThe nicotinic acetylcholine receptor (AChR) in adult skeletal muscle is composed of α‐, β‐, ϵ‐, and δ‐sub‐units and is localized at the neuromuscular junction; in contrast, the more diffusely distributed fetal form is composed of α‐, β‐, γ‐, and δ‐subunits. To define sequences necessary for the transcriptional regulation of the mouse ϵ‐subunit gene, we sequenced and analyzed 1036 bp upstream of the transcription start site. Using deletion analysis of the 5′‐flanking region linked to the bacterial chloramphenicol acetyltransferase (CAT) gene and transfection of the resulting constructs into established cell lines, we demonstrate that a 151 bp fragment exhibits cell type‐ and differentiation‐specific promoter activity. This activity was independent of a myogenic factor putative binding site (E‐box). However, transactivation experiments with recombinant myoD, myogenin, or MRF4 showed that the E‐box was functional and that MRF4 preferentially transactivates the ϵ‐promoter. Thus, like other AChR promoters, the proximal region of the ϵ‐promoter contains information for cell type‐specific and developmental regulation of CAT and can be transactivated by myogenci factors in cultured cell lines. Unlike the other AChR promoters characterized to date, ϵ‐promoter function can be partially independent of myogenic factors of the helix‐loop‐helix class. © 1993 Wiley‐Liss, Inc.