The binding of pyrophosphate to myosin, heavy meromyosin, and Subfragment 1 was studied by an equilibrium dialysis technique. Myosin binds approximately 2 (1.82) moles/5 × 105 g of protein, K = 2.07 × 106. Identical results were obtained with myosin prepared by the ammonium sulfate precipitation procedure with or without LiCl and the dilution method. Red and white skeletal muscle myosin bound the same number of moles of pyrophosphate, and variation of the ionic strength up to 0.6 did not affect either the binding number or the binding constant. No selective blocking of any of the sites could be achieved with N-ethylmaleimide or p-chloromercuribenzoate. Heavy meromyosin bound 1.83 moles of pyrophosphate per 3.6 × 105 g, and Subfragment 1 with a molecular weight of 9.74 × 104 to 1.19 × 105 bound 0.85 mole/105 g, K = 3.67 × 106. Natural actomyosin bound only 1 mole of pyrophosphate per 5 × 105 g of myosin. The addition of actin to myosin reduced both the number of binding sites and the affinity constant. The effect of actin on the pyrophosphate binding of heavy meromyosin was greater than on myosin and led to essentially complete suppression of binding. When heavy meromyosin was digested with trypsin (heavy meromyosin to trypsin, 16:1) at 23°, there was no alteration in the potassium-ethylenediaminetetraacetate activation or the calcium-activated ATPase activity. Isolated Subfragment 1 had a specific potassium-EDTA-activated ATPase activity which was 1.86 times higher than that of heavy meromyosin and a calcium-activated ATPase activity which was 2.60 times higher. After a 10-min tryptic digestion of heavy meromyosin, 25 to 45% of the total nitrogen was nonprecipitable with 6% trichloracetic acid. Disc electrophoresis of Subfragment 1 isolated by gel filtration on Sephadex G-200 contained two major bands and several minor ones in the leading edge of the peak, while the trailing edge contained only one major band and a few minor bands. The simplest explanation of these results is that tryptic digestion of heavy meromyosin produces two subfragments, each of which binds pyrophosphate, hydrolyzes ATP, and has the ability to combine with actin.