Force generation in striated muscle is coupled with inorganic phosphate (P(i)) release from myosin, because force falls with increasing P(i) concentration ([P(i)]). However, it is unclear which steps in the cross-bridge cycle limit loaded shortening and power output. We examined the role of P(i) in determining force, unloaded and loaded shortening, power output, and rate of force development in rat skinned cardiac myocytes to discern which step in the cross-bridge cycle limits loaded shortening. Myocytes (n = 6) were attached between a force transducer and position motor, and contractile properties were measured over a range of loads during maximal Ca(2+) activation. Addition of 5 mM P(i) had no effect on maximal unloaded shortening velocity (V(o)) (control 1.83 +/- 0.75, 5 mM added P(i) 1.75 +/- 0.58 muscle lengths/s; n = 6). Conversely, addition of 2.5, 5, and 10 mM P(i) progressively decreased force but resulted in faster loaded shortening and greater power output (when normalized for the decrease in force) at all loads greater than approximately 10% isometric force. Peak normalized power output increased 16% with 2.5 mM added P(i) and further increased to a plateau of approximately 35% with 5 and 10 mM added P(i). Interestingly, the rate constant of force redevelopment (k(tr)) progressively increased from 0 to 10 mM added P(i), with k(tr) approximately 360% greater at 10 mM than at 0 mM added P(i). Overall, these results suggest that the P(i) release step in the cross-bridge cycle is rate limiting for determining shortening velocity and power output at intermediate and high relative loads in cardiac myocytes.