To define transcriptional control elements responsible for muscle-specific expression of the human myoglobin gene, we performed mutational analysis of upstream sequences (nucleotide positions -373 to +7 relative to the transcriptional start site) linked to a firefly luciferase gene. Transient expression assays in avian and mammalian cells indicated that a CCCACCCCC (CCAC box) sequence (-223 to -204) is necessary for muscle-specific transcription directed either by the native myoglobin promoter or by a heterologous minimal promoter linked to the myoglobin upstream enhancer region. A putative MEF2-like site (-160 to -169) was likewise necessary for full transcriptional activity in myotubes. Mutations within either of two CANNTG (E-box) motifs (-176 to -148) had only minimal effects on promoter function. We identified and partially purified from nuclear extracts a 40-kDa protein (CBF40) that binds specifically to oligonucleotides containing the CCAC box sequence. A mutation of the CCAC box that disrupted promoter function in vivo also impaired binding of CBF40 in vitro. These data suggest that cooperative interactions between CBF40 and other factors including MEF-2 are required for expression of the human myoglobin gene in skeletal muscle.