Elevated levels of phosphate (Pi) and decreased pH (i.e. acidosis) are believed to contribute to muscular fatigue by directly inhibiting muscle's molecular motor, myosin. However, the mechanisms of inhibition are not fully understood at a molecular level. We determined the effects of elevated Pi (30mM) and low pH (6.5) at saturating ATP (2mM), separately and in combination, on myosin's ability to move actin in an in vitro motility assay (Vactin). Decreasing the pH without added Pi significantly (p<0.05) slowed Vactin (6.1 ± 0.2 at pH 7.4 vs. 1.6 ± 0.2 um/s at pH 6.5). The addition of Pi slightly increased Vactin at pH 7.4 (6.1 ± 0.2 at 0mM Pi vs. 6.8 ± 0.2 um/s at 30mM Pi), while at pH 6.5, Pi increased Vactin ~2‐fold (1.6 ± 0.2 at 0mM Pi vs. 3.3 ± 0.2 um/s at 30mM Pi). Repeating these experiments at sub‐saturating ATP showed that elevated Pi decreased Vactin below 1mM ATP at pH 7.4. However, at pH 6.5, elevated Pi continued to increase Vactin at all measured ATP concentrations (from 100uM to 2mM). These data indicate that the effects of Pi on myosin depend on both the pH and the ATP concentration. Since ATP affects the rigor lifetime and pH is believed to affect the ADP‐lifetime, these data imply that Pi may be able to rebind to myosin at more than one step in the cross‐bridge cycle. This work was funded by American Heart Association SDG grant (09SDG2260776) to Edward P. Debold