We have shown that myosin light chain phosphorylation inhibits fiber shortening velocity at high temperatures, 30° C, in the presence of the phosphate analog, vanadate. Vanadate inhibits tension by reversing the transition into force generating states, thus mimicking a pre-power stroke state. We have previously shown that at low temperatures vanadate also inhibits velocity, but at high temperatures it does not, with an abrupt transition in inhibition occurring near 25°C (Pate et al, Biophys. J. 66:1554, 1994). Here we show that for fibers activated in the presence of 0.5 mM vanadate, at 30°C, shortening velocity is not inhibited in dephosphorylated fibers, but is inhibited by 37 ±10% in fibers with phosphorylated myosin light chains. There is no effect of phosphorylation on fiber velocity in the presence of vanadate at 10°C. The k_m for ATP, defined by the maximum velocity of fibers partially inhibited by vanadate at 30°C, is 20 ± 4 µM for phosphorylated fibers and 192 ± 40 µM for dephosphorylated fibers, showing that phosphorylation also affects the binding of ATP. Fiber stiffness is not affected by phosphorylation. Inhibition of velocity by phosphorylation at 30°C depends on the phosphate analog, with approximately 12% inhibition in fibers activated in the presence of 5 mM BeF₃ and no inhibition in the presence of 0.25 mM AlF₄. Our results show that myosin phosphorylation can inhibit shortening velocity in fibers with large populations of myosin heads trapped in pre-power stroke states, such as occurs during muscle fatigue.