The muscle-specific, basic helix-loop-helix transcription factor MyoD can induce cells from other mesenchymal lineages to express a skeletal muscle phenotype. Interestingly, MyoD is initially upregulated in myogenic cells incubated with bone morphogenetic proteins (BMPs), a treatment that induces osteogenic differentiation, suggesting that MyoD has a role in BMP-induced osteogenesis of myogenic cells. This possibility is supported by our observations that muscle satellite cells derived from adult MyoD-/- mice show severely impaired osteogenic induction by BMP-7 (osteogenic protein 1; OP-1) as indicated by the decreased gene expression of the bone markers alkaline phosphatase, osteocalcin, Runx2/Cbfa1, and Osterix. Ectopic expression of MyoD increased alkaline phosphatase activity and Osterix mRNA expression in response to BMP treatment. Similarly, ectopic expression of MyoD in the pluripotent mesenchymal cell line C3H10T1/2 increased alkaline phosphatase activity induced by BMP-7. Transcription assays showed that transfection with a MyoD-expression vector, but not other myogenic basic helix-loop-helix transcription factors (Myf5, myogenin) increased Runx2/Cbfa1 transactivation of a reporter gene construct containing either six OSE sequences in tandem or a single OSE site. This effect was enhanced by BMP treatment. These studies, therefore, demonstrate that the muscle transcription factor MyoD is required for efficient BMP-induced osteogenesis of myogenic cells and indicate that MyoD might exert its effects through co-operative interactions with Runx2/Cbfa1.