Duchenne muscular dystrophy (DMD) is a genetic disease characterized by skeletal muscle wasting and atrophy. Recent evidence suggests that the impaired skeletal muscle performance in DMD is not solely dependent on a loss of contractile muscle mass. In this study the myosin motor function of mdx and control (wildtype, WT) mice was compared using pure myosin isoforms in an "in vitro motility assay" (IVMA). Actin sliding velocity (Vf) on myosin 2B extracted from single muscle fibers of gastrocnemius muscles was significantly lower in mdx mice (3.48 +/- 0.13 microm/s, n = 18) than in WT mice (4.02 +/- 0.19 microm/s, n = 10). No difference in Vf was found between myosin 1 extracted from soleus muscles of mdx (0.84 +/- 0.04 microm/s, n = 13) and of WT (0.89 +/- 0.04 microm/s, n = 10). The results suggest that the dystrophic process alters myosin molecular function, and this contributes to the functional impairment in dystrophic muscles. Muscle Nerve 40: 249-256, 2009.