Inhibition of muscle force development by acidic pH is a well known phenomenon, yet the exact mechanism by which a decrease in pH inhibits the Ca2+-activated force in striated myofilaments remains poorly understood. Whether or not the deactivation by acidic pH involves direct competition between Ca2+ and protons for regulatory binding sites on fast skeletal troponin C (TnC) or whether other proteins in thin filament regulation are important remains unclear. We measured the effects of acidic pH on Ca2+-dependent fluorescent changes in TnC labeled with the probe danzylaziridine (Danz), which reports Ca2+ binding to the regulatory (Ca2+-specific) sites. Measurements were also made with TnCDanz complexed with the inhibitory Tn unit, TnI, and in the whole Tn complex. Our results show that a drop in pH from 7.0 to 6.5 is associated with a 1.6-fold increase in the midpoint for the relation between free Ca2+ and Ca2+ binding to the regulatory sites on TnCDanz. However, when TnCDanz was present in its complex with either TnI alone or with TnI-TnT, the increase in midpoint free Ca2+ was increased by 3.5-fold. We tested whether this potentiation in the effect of acidic pH on Ca2+ binding to TnC is due to a pH-induced alteration in the binding of TnI to TnC. A decrease in pH from 7.0 to 6.5 was associated with a halving of the affinity of TnI for TnC. We also probed the effect of acidic pH on TnI. This was done (i) by measuring the intrinsic fluorescence of tryptophan residues in TnI alone and (ii) by measuring fluorescence of TnI (in the Tn complex) labeled at Cys-133 with 5-iodoacetamidofluorescein. A drop in pH from 7.0 to 6.5 was associated with a 15% decrease in intrinsic fluorescence and with a 30% decrease in the fluorescence of the 5-iodoacetamidofluorescein probe. We conclude, therefore, that while protons and Ca2+ may directly affect Ca2+ binding to regulatory sites on fast skeletal TnC, the effect of acidic pH on TnC Ca2+ binding is amplified in the TnI-TnC and Tn complexes by a pH-related effect on the affinity of TnI for TnC.