We show that DNA fragments, which span the skeletal muscle alpha-actin promoter, form specific complexes with proteins from nuclear extracts of 11 different cultured cell types. These include two myocyte stage specific cell types, myoblast and myotube. The myocyte nuclear extracts are distinguished from all of the others by the electrophoretic mobility of the predominant DNA complex. This complex is greatly diminished in the nine non-myocyte cell types, where a different complex predominates. The major myocyte and non-myocyte complexes are caused by distinct binding activities that compete for the same DNA sequence element. DNA footprint analysis shows that this element is located 78 nucleotides upstream of the transcription start site, within a region that appears essential for expression in developing myocytes.