Myosin was isolated from normal human skeletal muscle. After purification, there was virtually no actin, adenylate deaminase, or myokinase. Ribonucleoprotein was more tenacious than in preparations of rabbit myosin but could be eliminated by chromatography on diethylaminoethyl cellulose. The enzymatic properties of myosin from human skeletal muscle were more like that from red muscle than white muscle. Techniques were developed for electrophoresis of myosin in pyrophosphate buffer on agarose and 4% acrylamide gels. Immunodiffusion analysis, using antisera prepared to normal myosin, and electrophoretic methods were sensitive enough to detect differences between myosins from some species and from human heart and uterus. However, there was no difference between myosin from normal human muscle and the protein from patients with the major forms of muscular dystrophy (Duchenne, facioscapulohumeral, limb-girdle, myotonic).