Myosin from distal cruralis bundle of triceps femoris composed of slow-tonic and fast-twitch fibers in an about 1 to 1 ratio, from rectus abdominis containing a lower proportion of slow-tonic fibers, and from a fast-twitch sartorius muscle of the frog, was characterized by means of polyacrylamide gel electrophoresis in the presence of sodium dodecylsulphate (SDS), electrophoresis in non-dissociating conditions, and determination of the ATPase activity. SDS-polyacrylamide gel electrophoresis failed to reveal the presence of any specific light chain in myosin from slow-tonic fibers. Light chains having the same mobilities as fast-twitch LC1 and LC2 were observed; as previously described by Focant and Reznik [15], myosin from tonic fibers appears, on the other hand, devoid of LC3. By electrophoresis in non-dissociating conditions myosin from sartorius muscle was resolved into three components which comigrated with the three myosin isoenzymes from the fast-twitch posterior latissimus dorsi muscle of the chicken. Preparations from rectus abdominis and from cruralis bundle were shown to contain an additional component of lower electrophoretic mobility. Its relative proportion in these two muscles suggests that it represents the slow-tonic fiber myosin isoenzyme. Analysis of proteolytic digestion patterns revealed differences in the heavy chain structure of the isoenzymes from slow and fast fibers, respectively. As indicated by ATPase measurements, fast-twitch myosin exhibits a higher catalytic activity than myosin from slow-tonic fibers, the difference being of the same order as that reported for myosins from slow and fast muscles of higher vertebrates.