Nonenergized Streptococcus bovis cells, which were washed in potassium-phosphate buffer and incubated in Tris buffer containing 200 mm potassium chloride (pH 6.5), did not take up tetraphenylphosphonium ion (TPP+), but the same cells took up TPP+ when they were incubated in Tris buffer lacking potassium. This result indicated that passive potassium diffusion was creating an electrical potential (DELTAPSI) across the cell membrane. Neither cells took significant amounts of 9-aminoacridine (9-AA), an intracellular pH marker. Cells that were incubated in Tris buffer and treated with carbonyl cyanide m-chlorophenylhydrazone (CCCP) took up 9-AA, and this result indicated that this protonophore was facilitating proton influx. The ionophores monensin and lasalocid also caused 9-AA uptake, and it appeared that they were responsible for or responsive to potassium/proton antiport. However, there was also a rapid accumulation of 9-AA when the cells were treated with valinomycin, a potassium uniporter that cannot translocate protons. This latter result indicated that potassium efflux was associated with another avenue of proton influx (e.g., potassium/proton symport). Because cells treated with dicyclohexyl carbodiimide (DCCD) also exhibited valinomycin-dependent 9-AA uptake, it is unlikely that the F1F0ATPase or ATP formation was responsible for proton flux across the cell membrane.