CDO, a member of the Ig/fibronectin type III repeat subfamily of transmembrane proteins that includes the axon guidance receptor Robo, was identified by virtue of its down-regulation by the ras oncogene. We report here that one prominent site of cdo mRNA expression during murine embryogenesis is the early myogenic compartment (newly formed somites, dermomyotome and myotome). CDO is expressed in proliferating and differentiating C2C12 myoblasts and in myoblast lines derived by treating 10T1/2 fibroblasts with 5-azacytidine, but not in parental 10T1/2 cells. Overexpression of CDO in C2C12 cells accelerates differentiation, while expression of secreted soluble extracellular regions of CDO inhibits this process. Oncogenic Ras is known to block differentiation of C2C12 cells via downregulation of MyoD. Reexpression of CDO in C2C12/Ras cells induces MyoD; conversely, MyoD induces CDO. Reexpression of either CDO or MyoD rescues differentiation of C2C12/Ras cells without altering anchorage-independent growth or morphological transformation. CDO and MyoD are therefore involved in a positive feedback loop that is central to the inverse relationship between cell differentiation and transformation. It is proposed that CDO mediates, at least in part, the effects of cell-cell interactions between muscle precursors that are critical in myogenesis.