The proton ejection coupled to electron flow from succinate and/or endogenous substrate(s) to cytochrome c using the impermeable electron acceptor ferricyanide is studied in tightly coupled mitochondria isolated from two strains of the yeast Saccharomyces cerevisiae. (1) The observed H + ejection/2e − ratio approaches an average value of 3 when K + (in the presence of valinomycin) is used as charge-compensating cation. (2) In the presence of the proton-conducting agent carbonyl cyanide m-chlorophenylhydrazone, an H + ejection/2e − ratio of 2 is observed. (3) The low stoichiometry of 3H + ejected (instead of 4) per 2e − and the high rate of H + back-decay (0.1615 lnδ- ( ngatom) H + s and a half-time of 4.6 s for 10 mg protein) into the mitochondrial matrix are related to the presence of an electroneutral K +/H + antiporter which is demonstrated by passive swelling experiments in isotonic potassium acetate medium.