Corn (Zea mays L. cv Trojan T929) coleoptile membranes were fractionated on isopycnic sucrose density gradients. Two peaks of ATP-driven H(+)-transport activity, corresponding to the previously characterized tonoplast (1.07 grams per cubic centimeter) and Golgi (1.13 grams per cubic centimeter) fractions (Chanson and Taiz, Plant Physiol 1985 78: 232-240) were localized. Coincident with these were two peaks of inorganic pyrophosphate (PPi)-driven H(+)-transport. At saturating (3 millimolar) concentrations of Mg(2+):ATP, the rate of proton transport was further enhanced by the addition of 3 millimolar PPi, and the stimulation was additive, i.e. equal to the sum of the two added separately. The specific PPi analog, imidodiphosphate, antagonized PPi-driven H(+)-transport, but had no effect on ATP-driven transport. Moreover, PPi-dependent proton transport in both tonoplast-enriched and Golgi-enriched fractions was strongly promoted by 50 millimolar KNO(3), unlike the ATP-dependent H(+)-pumps of the same membranes. Taken together, the results indicate that PPi-driven proton transport is mediated by specific membrane-bound H(+)-translocating pyrophosphatases. Both potassium and a permanent anion (NO(3) (-) > Cl(-)), were required for maximum activity. The PPi-driven proton pumps were totally inhibited by N,N'-dicyclohexylcarbodiimide, but were insensitive to 100 millimolar vanadate. The PPi concentration in coleoptile extracts was determined using an NADH oxidation assay system coupled to purified pyrophosphate:fructose 6-phosphate 1-phosphotransferase (EC 2.7.1.90). The total pyrophosphate content of corn coleoptiles was 20 nanomoles/gram fresh weight. Assuming a cytoplasmic location, the calculated PPi concentration is sufficient to drive proton transport at 20% of the maximum rate measured in vitro for the tonoplast-enriched fraction, and 10% of the maximum rate for the Golgi-enriched fraction.