The plasma membrane V-ATPase of Manduca sexta larval midgut is an electrogenic proton pump located in goblet cell apical membranes (GCAM); it energizes, by the voltage component of its proton motive force, an electrophoretic K⁺/nH⁺ antiport and thus K⁺ secretion (Wieczorek, H., Putzenlechner, M., Zeiske, W., and Klein, U.(1991) J. Biol Chem. 266, 15340-15347). Midgut transepithelial voltage, indicating net active K⁺ transport, was found to be more than 100 mV during intermoult stages but was abolished during moulting. Simultaneously, ATP hydrolysis and ATP-dependent proton transport in GCAM vesicles were found to be reduced to 10-15% of the intermoult level. Immunocytochemistry of midgut cryosections as well as SDS-polyacrylamide gel electrophoresis and immunoblots of GCAM demonstrated that loss of ATPase activity paralleled the disappearance of specific subunits. The subunits missing were those considered to compose the peripheral V₁ sector, whereas the membrane integral V₀ subunits remained in the GCAM of moulting larvae. The results provide, for the first time, evidence that a V-ATPase activity can be controlled in vivo by the loss of the peripheral V₁ domain.