Abstract: A novel gene, Inhibitor of MyoD Family (I-mf), is provided which encodes novel proteins, I-mfa, I-mfb and I-mfc, involved in regulation of myogenesis during vertebrate development. I-mf is highly expressed in the sclerotome of developing vertebrates and is postulated to play an important role in patterning of the somite and determination sclerotomal cell fate. A unique, C-terminal interactional domain of the I-mf protein mediates physical interactions between I-mfa and members of the MyoD family of transcriptional activators and functions to inhibit transactivation of muscle specific genes by MyoD family members, thereby repressing myogenesis. Further characterization of I-mf activity shows that I-mf associates with MyoD family member proteins and retains them in the cytoplasm by masking their nuclear localization signals.