Abstract: H(+) transport driven by V H(+)-ATPase was found in membrane fractions enriched with ER/PM and Golgi/Golgi-like membranes of Saccharomyces cerevisiae efficiently purified in sucrose density gradient from the vacuolar membranes according to the determination of the respective markers including vacuolar Ca(2+)-ATPase, Pmc1::HA. Purification of ER from PM by a removal of PM modified with concanavalin A reduced H(+) transport activity of P H(+)-ATPase by more than 75% while that of V H(+)-ATPase remained unchanged. ER H(+) ATPase exhibits higher resistance to bafilomycin (I(50)=38.4 nM) than Golgi and vacuole pumps (I(50)=0.18 nM). The ratio between a coupling efficiency of the pumps in ER, membranes heavier than ER, vacuoles and Golgi is 1.0, 2.1, 8.5 and 14 with the highest coupling in the Golgi. The comparative analysis of the initial velocities of H(+) transport mediated by V H(+)-ATPases in the ER, Golgi and vacuole membrane vesicles, and immunoreactivity of the catalytic subunit A and regulatory subunit B further supported the conclusion that V H(+)-ATPase is the intrinsic enzyme of the yeast ER and Golgi and likely presented by distinct forms and/or selectively regulated.