Muscle fatigue from intense contractile activity is thought to result, in large part, from the accumulation of inorganic phosphate (P(i)) and hydrogen ions (H(+)) acting to directly inhibit the function of the contractile proteins; however, the molecular basis of this process remain unclear. We used an in vitro motility assay and determined the effects of elevated H(+) and P(i) on the ability of myosin to bind to and translocate regulated actin filaments (RTF) to gain novel insights into the molecular basis of fatigue. At saturating Ca(++), acidosis depressed regulated filament velocity (V(RTF)) by ≈ 90% (6.2 ± 0.3 vs. 0.5 ± 0.2 μm/s at pH 7.4 and 6.5, respectively). However, the addition of 30 mM P(i) caused V(RTF) to increase fivefold, from 0.5 ± 0.2 to 2.6 ± 0.3 μm/s at pH 6.5. Similarly, at all subsaturating Ca(++) levels, acidosis slowed V(RTF), but the addition of P(i) significantly attenuated this effect. We also manipulated the [ADP] in addition to the [P(i)] to probe which specific step(s) of cross-bridge cycle of myosin is affected by elevated H(+). The findings are consistent with acidosis slowing the isomerization step between two actomyosin ADP-bound states. Because the state before this isomerization is most vulnerable to P(i) rebinding, and the associated detachment from actin, this finding may also explain the P(i)-induced enhancement of V(RTF) at low pH. These results therefore may provide a molecular basis for a significant portion of the loss of shortening velocity and possibly muscular power during fatigue.