A method is described for the preparation of high purity myosin from small amounts of cardiac muscle. The method employs homogenization and prolonged extraction of the cardiac tissue. Purification is achieved through three successive precipitation-dissolution cycles and without the use of column chromatographic techniques. Purity of the myosin preparation is assessed at various stages of the purification procedure by sodium dodecylsulfate-acrylamide gel electrophoresis and by measurement of RNA and nucleoprotein content. With 1.5-2.0 g of rabbit right ventricle as the starting tissue, this method yields 4-6 mg myosin per g wet tissue. The method is also shown to give similar results with rabbit right ventricles hypertrophied by pulmonary stenosis.