A rapid procedure for isolating subfragment one (SF1) from myosin was found. SF1 can be isolated specifically from proteolytic digests of myosin in the presence of a millimolar concentration of magnesium chloride. Under such ionic conditions all of the rod portion and undigested myosin is selectively precipitated. A nucleotide trapping experiment indicated how important quick preparation of SF1 is for maintaining the active site structure. This method can also be utilized in the preparation of heavy meromyosin.