Intracellular acidosis is a putative agent of skeletal muscle fatigue, in part, because acidosis depresses the calcium (Ca2+) sensitivity and force production of muscle (18, 50). However, the molecular mechanisms behind this depression in Ca2+ sensitivity and force production are unknown. This gap in knowledge poses a significant challenge in generating a complete understanding of the fatigue process. To close this gap, the ability of myosin to bind to a single actin filament was measured under acidic conditions, in a laser trap assay, with and without regulatory proteins. Decreasing pH from 7.4 to 6.5 reduced the frequency of single actomyosin binding events at submaximal (pCa 8 – pCa 6), but not at maximal (pCa 5 - 4) concentrations. To delineate whether this was due to a direct effect on myosin versus an indirect effect on the regulatory proteins, troponin (Tn) and tropomyosin (Tm), binding frequency was also quantified in the absence of Tn and Tm. Acidosis did not alter the frequency of actomyosin binding events in the absence of regulatory proteins (1.4 ± 0.05 vs 1.4 ± 0.13 events/sec for pH 7.4 and 6.5). Additionally, acidosis did not significantly affect the size of myosin’s powerstroke, or the duration of binding events in the presence of regulatory proteins, at every pCa. These data suggest that acidosis impedes activation of the thin filament by competitively inhibiting Ca2+ binding to TnC. This slows the rate at which myosin initially attaches to actin, therefore less cross-bridges will be bound and generating force at any given sub-maximal pCa.