Betaglycan is a membrane-anchored proteoglycan coreceptor that binds transforming growth factor ␤ (TGF-␤) via its core protein and basic fibroblast growth factor through its glycosaminoglycan chains. In this study we evaluated the expression of betaglycan during the C 2 C 12 skeletal muscle differentiation. Betaglycan expression, as determined by Northern and Western blot, was up-regulated during the conversion of myoblasts to myotubes. The mouse betaglycan gene promoter was cloned, and its sequence showed putative binding sites for SP1, Smad3, Smad4, muscle regulatory factor elements such as MyoD and MEF2, and retinoic acid receptor. Transcriptional activity of the mouse betaglycan promoter reporter was also up-regulated in differentiating C 2 C 12 cells. We found that MyoD, but not myogenin, stimulated this transcriptional activity even in the presence of high serum. Betaglycan promoter activity was increased by RA and inhibited by the three isoforms of TGF-␤. On the other hand, basic fibroblast growth factor, BMP-2, and hepatocyte growth factor/scatter factor, which are inhibitors of myogenesis, had little effect. In myotubes, up-regulated betaglycan was also detectable by TGF-␤ affinity labeling and immunofluorescence microscopy studies. The latter indicated that betaglycan was localized both on the cell surface and in the ECM. Forced expression of betaglycan in C 2 C 12 myoblasts increases their responsiveness to TGF-␤2, suggesting that it performs a TGF-␤ presentation function in this cell lineage. These results indicate that betaglycan expression is up-regulated during myogenesis and that MyoD and RA modulate its expression by a mechanism that is independent of myogenin.Skeletal muscle myoblasts are the precursors to skeletal muscle fibers. During development these cells are maintained in a proliferative and undifferentiated state until appropriate signals cause them to undergo conversion into multinucleated myotubes.A network of muscle regulatory factors (MRFs), 1 some of which have been identified and characterized, governs this process. When myogenesis begins, myogenic regulatory genes encoding factors of the MyoD family are activated (myogenin, MRF4, and MRF5). These factors bind to specific DNA consensus sites called E boxes, which function as transcriptional enhancers of muscle differentiation genes (for review see Ref.