SUMMARY Single fibers, 25-40 μm wide and 0.5-0.7 mm long, were isolated from the flexor digitorum brevis muscle of the mouse. Force and movement were recorded(21-27°C) from the fiber as a whole and, in certain experiments, from a short marked segment that was held at constant length by feedback control. The maximum tetanic force, 368±57 kN/m2 (N=10), was not significantly different from that recorded in frog muscle fibers at equal temperature. However, the rising phase of the tetanus was considerably slower in the mammalian fibers, 202±20 ms (N=17) being required to reach 90% of maximum tetanic force as compared with 59±4 ms(N=20) in the frog muscle fibers. Similar to the situation in frog muscle fibers, the force-velocity relation exhibited two distinct curvatures located on either side of a breakpoint near 80% of the isometric force. Maximum speed of shortening was 4.0±0.3 fiber lengths s-1(N=6). The relationship between tetanic force and sarcomere length was studied between 1.5 and 4.0 μm sarcomere spacings, based on length-clamp recordings that were free of `tension creep'. There was a flat maximum (plateau) of the length-tension relation between approximately 2.0 and 2.4 μm sarcomere lengths. The descending limb of the length-tension relation (linear regression) intersected the length axis (zero force) at 3.88μm and reached maximum force at 2.40 μm sarcomere length. The slope of the descending limb is compatible with a thick filament length of 1.63 μm and an average thin filament length of 1.10 μm. These values accord well with recent electron microscope measurements of myofilament length in mammalian muscle.