The product of the proto-oncogene Jun inhibits myogenesis. Constitutive expression of Jun in myoblasts interferes with the expression and the function of MyoD protein. In transient transfection assays Jun inhibits transactivation of the MyoD promoter, the muscle creatine kinase enhancer, and a reporter gene linked to MyoD DNA-binding sites. Conversely, MyoD suppresses the transactivation by Jun of genes linked to an AP-1 site. We demonstrate that both in vivo and in vitro MyoD and Jun proteins physically interact. Mutational analysis suggests that this interaction occurs via the leucine zipper domain of Jun and the helix-loop-helix region of MyoD.