The fluorescence quenching of 9-aminoacridine (9AA) in suspension of beef heart EDTA submitochondrial particles was studied and was used to calculate the pH gradient between these particles and the medium. This pH gradient, which is energy dependent, is also dependent strongly on the presence of anion species in the medium. It is 2.2 pH units in acetate medium and can be as high as 3.6 units in the presence of other highly lyophilic anions. The anions tested were found to be effective in the following order: SCN- greater than I- greater than NO3- greater than Br- greater than Cl-. The validity of the deltapH calculations was confirmed by comparison with deltapH values calculated from NH4+ uptake. In contrast, calculations based on quinacrine (QA) fluorescence quenching under the same assumption used for 9AA did not agree with NH4+ measurements and show quantitative and in some cases even qualitative differences. Both carbonyl cyanide p-trifluoromethoxyphenylhydrazone and NH4+ decreased deltapH significantly. When the rate of electron transport is slow, i.e., with succinate as substrate or with NADH and low concentration of rotenone, very low concentration of nigericin (less than 20 ng/ml) decreased deltapH. Under these conditions, valinomycin antagonized the nigericin effect and restored deltapH to its original value. Upon increasing nigericin concentration (greater than 100 ng/ml) the valinomycin effect is gradually replaced by a slower response of further reduction of deltapH.