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Alternatively, various subunits of the V-ATPase, including subunits a, d, A, and C, have been shown to control the activity of the V-ATPase by modulating the coupling of proton transport to ATP hydrolysis. The presence of various isoforms of subunits a, d, and C in clear cells may, therefore, reflect different levels of V-ATPase activity in the membrane domains and intracellular compartments in which they are expressed. We have previously shown in rat and mouse epididymis that clear cells express both the B1 and the B2 isoform. While Bl completely colocalized with E2 in apical microvilli and subapical vesicles, B2 was located only in E2-positive vesicles and was absent from microvilli. Coimmunoprecipitation assays from kidney extracts showed that C1, G1, and E2 interact preferentially with B2 and that C2b, G3, and E2 interact with Bl. Out of the V0 domain subunits, dl interacts with both Bl and B2, whereas d2 associates with B1 exclusively. Subunits al, a2, and a4 all bind to B2, but only a4 can associate with Bl. The localization of B2, therefore, correlates with the present localization of isoforms al, dl, Cl, and G1 in subapical vesicles and their absence in apical microvilli. However, the absence of a2 from subapical vesicles, where B2 is located, was not expected. Abundant al and a4 was detected in these vesicles and might compete away interaction between B2 and a2 in this compartment.