Mycoplasma gallisepticum cells incubated in 250 mM NaCl solutions in the absence of glucose showed a progressive fall in intracellular ATP concentration over a period of 2 to 3 h. When the ATP level fell below 40 microM the cell began to swell and become progressively permeable to [14C]inulin and leak intracellular protein and nucleotides. The addition of nondiffusable substances such as MgSO4 or disaccharides prevented swelling, suggesting that NaCl (and water) entry was due to Gibbs-Donnan forces. The addition of glucose after the initiation of cell swelling increased intracellular ATP, induced cell shrinkage, and prevented the release of intracellular components. The ATPase inhibitor dicyclohexylcarbodiimide, which collapsed the chemical and electrical components of the proton motive force, caused rapid cell swelling in the presence of glucose (and high intracellular ATP levels). Extracellular impermeable solutes such as MgSO4 and disaccharides prevented swelling of dicyclohexylcarbodiimide-treated cells incubated in NaCl. It was postulated that Na+ that diffused into the cell was extruded by an electrogenic Na+-H+ exchange (antiport) energized by the proton motive force established by the dicyclohexylcarbodiimide-sensitive H+-ATPase.