Phosphate incorporation by the phosphorylatable light chains (P-LC) of myosin is associated with isometric twitch force potentiation in intact fast-twitch muscle. The purpose of this study was to examine the association between myosin P-LC phosphorylation and force potentiation at higher stimulation frequencies (1-150 Hz) using mouse extensor digitorum longus (EDL) muscles at 25 degrees C. Peak isometric force and the peak rate of isometric force development (+dF/dtmax) were measured at selected test frequencies before and after the application of a 5-Hz 20-s conditioning stimulation known to increase P-LC phosphate content. Associated with a ninefold elevation in myosin P-LC phosphate content (to 0.72 mol phosphate/mol P-LC), +dF/dtmax was increased at all test frequencies (mean 27%, range 20-37%). After the conditioning stimulus, peak isometric force was increased by approximately 15% for frequencies 1-15 Hz. However, at 20-150 Hz, the increase in +dF/dtmax was not associated with force potentiation, since peak force was diminished by 5-40%. These data reveal that the stimulation frequency limit for the potentiation of peak force production associated with myosin P-LC phosphorylation is < 20 Hz in mouse EDL at 25 degrees C. Furthermore, the data suggest that increases in the rate constant describing the rate of cross-bridge transition from a non-force-generating to a force-generating state mediated by myosin P-LC phosphorylation may be responsible for the general increase in +dF/dtmax and for the force potentiation at 1-15 Hz.