Electrogenic ATPase activity on the peribacteroid membrane from soybean (Glycine max L. cv Bragg) root nodules is demonstrated. Membrane energization was monitored using suspensions of intact peribacteroid membrane-enclosed bacteroids (peribacteroid units; PBUs) and the fluorescent probe for membrane potential (DeltaPsi), bis-(3-phenyl-5-oxoisoxazol-4yl) pentamethine oxonol. Generation of a positive DeltaPsi across the peribacteroid membrane was dependent upon ATP, inhibited by N,N'-dicyclohexyl-carbodiimide and vanadate, but insensitive to N-ethylmaleimide, azide, cyanide, oligomycin, and ouabain. The results suggest the presence of a single, plasma membrane-like, electrogenic ATPase on the peribacteroid membrane. The protonophore, carbonyl-cyanide m-chlorophenyl hydrazone, completely dissipated the established membrane potential. The extent of reduction in the steady state membrane potential upon addition of ions was used to estimate the relative permeability of the peribacteroid membrane to anions. By this criterion, the relative rates of anion transport across the peribacteroid membrane were: NO(3) (-) > NO(2) (-) > Cl(-) > acetate(-) > malate(-). The observation that 10 millimolar NO(3) (-) completely dissipated the membrane potential was of particular interest in view of the fact that NO(3) (-) inhibits symbiotic nitrogen fixation. The possible function of the ATPase in symbiotic nitrogen fixation is discussed.