The difference in kinetic properties between two myosin isozymes (V1 and V3) in rat ventricular myocardium was studied by determining the steady-state force-velocity (P-V) relations in the ATP-dependent movement of V1 and V3-coated polystyrene beads on actin cables of giant algal cells mounted on a centrifuge microscope. The maximum unloaded velocity of bead movement was larger for V1 than for V3. The velocity of bead movement decreased with increasing external load applied by the centrifuge microscope, and eventually reached zero when the load was equal to the maximum isometric force (P0) generated by the myosin heads. The maximum isometric force P0 was less than 10 pN, and did not differ significantly between V1 and V3. The P-V curves consisted of a hyperbolic part in the low force range and a non-hyperbolic part in the high force range. The critical force above which the curve deviated from the hyperbola was much smaller for V1 than for V3. An analysis using a model with an extremely small number of myosin heads involved in the bead movement suggested a marked difference in kinetic properties between V1 and V3.