ABSTRACT Achieving an understanding of the biosynthesis, assembly and intracellular targeting of the vacuolar H+-ATPase is critical for understanding the distribution of acidic compartments and the regulation of organelle acidification. The assembly of the yeast vacuolar H+-ATPase requires the attachment of several cytoplasmically oriented, peripheral subunits (the V1 sector) to a complex of integral membrane subunits (the Vo sector) and thus is not easily described by the established mechanisms for transport of soluble or vacuolar membrane proteins to the vacuole. In order to examine the assembly of the enzyme complex, yeast mutants lacking one of the subunit genes have been constructed and the synthesis and assembly of the other subunits have been examined. In mutants lacking one subunit, the remaining ATPase subunits seem to be synthesized, but in many cases are either not assembled or not targeted to the vacuole. Immunofluorescence and subcellular fractionation experiments have revealed that deletion of one peripheral subunit prevents the other peripheral subunits, but not the integral membrane subunits, from reaching the vacuole. In contrast, the absence of one of the integral membrane subunits appears to prevent both the peripheral subunits and another integral subunit from reaching the vacuole and also results in reduced cellular levels of the other integral membrane subunit. These data suggest that transport of integral and peripheral membrane subunits to the vacuole may employ somewhat independent mechanisms and that some assembly of the V1 and Vo sectors may occur before the two sectors are joined. Current models for the assembly process and the implications for organelle acidification are discussed.