The biosynthesis and assembly of the peripheral sector (V,) of the vacuolar protontranslocating adenosine triphosphatase (V-ATPase) was studied in a bovine kidney epithelial cell line. Monolayer cultures of cells were metabolically radiolabeled with Tran3'S-label and the V-ATPase subsequently immunoprecipitated using a monoclonal antibody raised against the bovine brain-coated vesicle proton pump. The V-ATPase immunoprecipitated from the bovine kidney cell line has a subunit composition very similar to that of the bovine brain-coated vesicle proton pump and the V-ATPase prepared from other kidney tissues. Radiolabeling the cells for increasing times showed that the V, or peripheral portion of the V-ATPase is assembled within 10-1 5 min; the intact V,V, complex is also detectable within 10-1 5 min. Fractionation of the cells into cytosolic and membrane components prior to immunoprecipitation revealed that there is a significant pool of V, in the cytosol; a similar complex is also found in bovine brain cytosol. Pulse-chase studies suggest that this cytosolic pool is not an obligate precursor for membranebound V,Vo and does not exchange with the membrane V, population at later times. N o qualitative differences in assembly were observed when pulse-chase studies were performed at 15ยฐC or in the presence of brefeldin A. This suggests that assembly of V,V, is probably completed in the endoplasmic reticulum prior to distribution of the enzyme throughout the cell, with a cytosolic pool of V, of unknown function existing in parallel with the fully assembled complex.