CD44 is a transmembrane protein that plays a role in cell–cell interactions and motility in a number of cell types. Cell–cell interactions are critical for myoblast differentiation and fusion but whether CD44 regulates myogenesis is unknown. Here, we show that CD44 plays a functional role in early myogenesis. Analyses of myofiber cross-sectional area, after local injury in mouse tibialis anterior (TA) muscles, revealed that growth was transiently delayed in the absence of CD44. A muscle-intrinsic role for CD44 is suggested as primary myoblasts from CD44−/− mice displayed attenuated differentiation and subsequent myotube formation at early times in a differentiation-inducing in vitro environment. Chemotaxis of CD44−/− myoblasts toward hepatocyte growth factor (HGF) and basic fibroblast growth factor (bFGF) was totally abrogated, although expression of their respective receptors did not appear to differ from wild-type. Furthermore, motility of CD44−/− myoblasts was decreased at early stages of differentiation as determined by time-lapse microscopy. Wild-type myoblasts contained two subpopulations of slow- and fast-migrating cells, whereas CD44−/− myoblasts were composed predominantly of the slower migrating subpopulation. Taken together, these data suggest that myoblast migration and differentiation are closely linked and CD44 is a key regulator. J. Cell. Physiol. 209: 314–321, 2006. © 2006 Wiley-Liss, Inc.