Metal-induced conformational changes in actin at 20 °C have been investigated as a function of pH using actin labeled at Cys-374 with N-(iodoacety1)-N'-(5-sulfo-1-naphthyl)ethylenediamine. At pH 8, the addition of a high Ca2+concentration (2 mM) to G-actin gives an instantaneous fluorescence increase while the addition of a high Mg2+concentration gives both an instantaneous and a slow fluorescence increase. The instantaneous increase is interpreted as divalent cation binding to low-affinity, relatively nonspecific sites, while the slow response is attributed to Mg2+binding to specific sites of moderate affinity [Zimmerle, C. T., Patane, K., & Frieden, C. (1987) Biochemistry 26, 6545–6552]. The magnitudes of both the instantaneous and slow fluorescence increases associated with Mg2+addition to G-actin are shown here to decrease as the pH is lowered while the fluorescence of labeled G-actin in the presence of low or moderate Ca2+concentrations (<200 μM) increases. The pH-dependent data suggest that protonation of a single class of residues with an approximate pK of 6.8 alters the immediate environment of the label differently depending upon the cation bound at the moderate-affinity site. The pH-dependent changes in the magnitude of the slow fluorescence response upon Mg2+addition to Ca2+-actin are not associated with changes in the Mg2+affinity at the moderate-affinity site but result from protonation altering the fluorescence response to Mg2+binding. Protonation of this same class of residues is proposed to induce an actin conformation similar to that induced by cation binding at the low-affinity sites. Ca2+binding at a single high-affinity (~10-9M) site remains quite tight, with the rate constant of release decreasing approximately 10-fold as the pH is lowered from 8 to 6.