1.1. When the fluorescent antibody method was first used to study the localization of myosin in the myofibrils of skeletal muscle, the observation of fluorescence in the A band of the sarcomere was thought to be in general agreement with prior conclusions that had been based on the differential removal of specific proteins from the myofibril by extraction procedures. The heterogeneity subsequently observed in preparations of antimyosin has been variously attributed to contamination of the immunizing antigen with AMP deaminase (AMP aminohydrolase, EC 3.5.4.6), or with actomyosin and nucleoprotein.2.2. Because the presence of heterologous antibodies in fluorescein-labeled antimyosin may give false or misleading information about the precise spatial distribution of myosin within the sarcomere, the myosin-antimyosin system has been reinvestigated to determine: (a) whether the heterogeneity of myosin is influenced by its method of preparation; (b) how this in turn affects the homogeneity of antisera to myosin; and (c) if the extraneous antigens are (random) fragments of myosin, or true contaminants.3.3. Myosin and actomyosin were prepared from chicken skeletal muscle, purified by two different methods, and used to produce antisera in rabbits. The three aspects of the problem were then examined by correlating the different methods of preparation with variations in the ultraviolet absorption spectra, AMP deaminase activities, ATPase (ATP phosphohydrolase, EC 3.6.1.3) activities and the immunochemical properties of the purified proteins.4.4. The results show that the heterogeneity of myosin is reduced by ammonium sulfate fractionation, that both myosin and its antisera remain heterogeneous, and that the heterogeneity is due to the presence of true contaminants in the purified antigen. 1. When the fluorescent antibody method was first used to study the localization of myosin in the myofibrils of skeletal muscle, the observation of fluorescence in the A band of the sarcomere was thought to be in general agreement with prior conclusions that had been based on the differential removal of specific proteins from the myofibril by extraction procedures. The heterogeneity subsequently observed in preparations of antimyosin has been variously attributed to contamination of the immunizing antigen with AMP deaminase (AMP aminohydrolase, EC 3.5.4.6), or with actomyosin and nucleoprotein. 2. Because the presence of heterologous antibodies in fluorescein-labeled antimyosin may give false or misleading information about the precise spatial distribution of myosin within the sarcomere, the myosin-antimyosin system has been reinvestigated to determine: (a) whether the heterogeneity of myosin is influenced by its method of preparation; (b) how this in turn affects the homogeneity of antisera to myosin; and (c) if the extraneous antigens are (random) fragments of myosin, or true contaminants. 3. Myosin and actomyosin were prepared from chicken skeletal muscle, purified by two different methods, and used to produce antisera in rabbits. The three aspects of the problem were then examined by correlating the different methods of preparation with variations in the ultraviolet absorption spectra, AMP deaminase activities, ATPase (ATP phosphohydrolase, EC 3.6.1.3) activities and the immunochemical properties of the purified proteins. 4. The results show that the heterogeneity of myosin is reduced by ammonium sulfate fractionation, that both myosin and its antisera remain heterogeneous, and that the heterogeneity is due to the presence of true contaminants in the purified antigen.