The question of whether Thiobacillus acidophilus maintains its cytoplasmic pH at values close to neutrality by active or passive means was explored by subjecting the organism to long-term starvation (up to 22 days). Starving cells maintained a delta pH of 2 to 3 U throughout starvation, although cellular poly-beta-hydroxybutyric acid and ATP, the proton motive force, and culture viability were low or not detectable after 200 h. Cells exposed to azide or azide plus N,N'-dicyclohexylcarbodiimide immediately exhibited characteristics of cells starved for more than 200 h. Thus, a large delta pH in T. acidophilus was maintained in the absence of ATP, ATPase activity, respiration, significant levels of proton motive force, and cell viability and was therefore not dependent on chemiosmotic ionic pumping. The transition from a metabolically active to an inactive state was accompanied by a large increase in the positive membrane potential, which nearly completely compensated for the delta pH in the inactive cells. The longevity of the acidophile during starvation was comparable to that reported previously for neutrophiles, and the loss of viability occurred not because of the acidification of the cytoplasm but apparently because of energy depletion.