Skeletal muscle force output is regulated through Ca(2+)-mediated alterations of the rate at which cross bridges make the transition from non-force-generating to force-generating states, defined by the rate constant fapp. In skinned-fiber models, phosphate incorporation by the regulatory light chain (R-LC) subunits of myosin increases fapp independent of Ca2+, thus increasing the Ca2+ sensitivity for the rate and extent of steady-state force development. The goal of this study was to determine whether phosphate incorporation by the R-LC subunits of skeletal muscle is related to the maximal rate of isometric force development (+dF/dtmax) in intact muscle. Changes in myosin phosphate content and contractile performance were analyzed at selected times after the application of a 5-Hz 20-s conditioning stimulus (CS) employed specifically to elevate R-LC phosphate content in mouse extensor digitorum longus at 25 degrees C. R-LC phosphate content (in mol phosphate/mol R-LC) increased from 0.13 +/- 0.04 at rest to 0.68 +/- 0.02 20 s after the CS and by 360 s after the CS R-LC phosphate content had decayed to 0.37 +/- 0.06. Values obtained for twitch and tetanic +dF/dtmax after the CS were strongly correlated to R-LC phosphate content (r = 0.97 and 0.96, respectively), suggesting that phosphate incorporation by skeletal myosin R-LC contributes to an enhanced rate of isometric force development in fast-twitch skeletal muscle.