Two studies were conducted to examine the effect of myosin regulatory light chain (R-LC) phosphorylation on the rate and extent of shortening in submaximally activated mouse extensor digitorum longus muscles in vitro at 25 degrees C. For each study, R-LC phosphate content was increased fivefold by application of a 5-Hz, 20-s conditioning stimulus (CS) to 0.65-0.68 mol phosphate/mol R-LC; this level was sustained between 10 and 40 s after the CS. Maximum isometric twitch force and the maximum rate of force development (+dF/dtmax) were potentiated in the range 13-17% and 9-17% (P < 0.05), respectively, after the CS. In study 1, the maximal rate and extent of shortening were significantly enhanced by 10 and 21% (P < 0.001), respectively, when measured using a twitch zero-load clamp technique. In study 2, the force-velocity and force-displacement relationships were both augmented when determined with the twitch afterload technique. Displacement was enhanced between 20 and 82% for loads that ranged from 3 to 75% of active peak twitch force, whereas velocity was increased 6-8% over the same range (P < 0.05), including the predicted maximum velocity (Vmax; 5.08 vs. 4.69 muscle length/s). In both studies the increase in velocity likely represents a shift along the force-velocity relationship toward true Vmax that reflects a decrease in relative load due to force potentiation. Furthermore, with the decrease in relative load, displacement at a given load was also increased. Potentiated displacement and extent of R-LC phosphorylation also decreased in parallel when studied for 5 min after the CS. The increase in muscle shortening is a novel finding and suggests a function for R-LC phosphorylation with respect to movement because both peak work and power were also enhanced by up to 22%. These effects are consistent with an R-LC phosphorylation-induced increase in fapp, the apparent rate constant that describes the cross-bridge transition from the non-force-generating to the force-generating state.