The isometric contractile properties of single myofibrils of rabbit skeletal muscle were studied at various sarcomere lengths. Single myofibrils were suspended between the tips of one rigid and one flexible glass microneedle, and their force production was determined by detecting the bending of the flexible microneedle photo-electronically. The active force vs. sarcomere length relation had an ascending limb (0.7-2.25 micron), a plateau (2.25-2.5 micron), and a descending limb (2.5-3.8 micron), which was similar to that of frog skeletal muscle. The passive force became increasingly apparent beyond a sarcomere length of 2.4 micron. These results can reasonably be explained based on the sliding filament mechanism by assuming the sarcomere geometry of rabbit muscle. The plateau, with a produced force of about 256 kN/m2, and the linear decline of force in the descending limb of the single myofibrils were essentially the same as those for frog muscle fibers. However, the slope of the force decline in the ascending limb was far steeper than that for frog muscle. This suggests that internal elements of sarcomeres are different between rabbit and frog muscles.