ABSTRACT The role of the actin filament‐associated protein nebulin on mechanical and kinetic properties of the actomyosin motor was investigated in skeletal muscle of wild‐type (ω t ) and nebulin‐deficient ( nebulin _/‐ ) mice that were 1 d old' an age at which sarcomeric structure is still well preserved. In Ca 2+ ‐activated skinned fibers from psoas muscle, we determined the Ca 2+ dependence of isometric force and stiffness, the rate of force redevelopment after unloaded shortening (k TR ), the power during isotonic shortening, and the unloaded shortening velocity (V 0 ). Our results show a 65% reduction in isometric force in nebulin ‐ /‐ fibers at saturating [Ca 2+ ], whereas neither thin‐filament length nor the Ca 2+ sensitivity of the contractile system is affected. Stiffness measurements indicate that the reduction in isometric force is due to a reduction in the number of actin‐attached myosin motors, whereas the force of the motor is unchanged. Furthermore, in nebulin ‐/‐ fibers, k TR is decreased by 57%, V 0 is increased by 63%, and the maximum power is decreased by 80%. These results indicate that, in the absence of nebulin, the attachment probability of the myosin motors to actin is decreased, revealing a direct role for nebulin in promoting strong actomyosin interactions responsible for force and power production.— Bang, M.‐L., Caremani, M., Brunello, E., Littlefield, R., Lieber, R. L., Chen, J., Lombardi, V., Linari, M. Nebu‐lin plays a direct role in promoting strong actin‐myosin interactions. FASEBJ. 23,4117‐4125 (2009). www.fasebj.org