All eukaryotic cells contain multiple acidic organelles, and V-ATPases are central players in organelle acidification. Not only is the structure of V-ATPases highly conserved among eukaryotes, but there are also many regulatory mechanisms that are similar between fungi and higher eukaryotes. These mechanisms allow cells both to regulate the pHs of different compartments and to respond to changing extracellular conditions. The Saccharomyces cerevisiae V-ATPase has emerged as an important model for V-ATPase structure and function in all eukaryotic cells. This review discusses current knowledge of the structure, function, and regulation of the V-ATPase in S. cerevisiae and also examines the relationship between biosynthesis and transport of V-ATPase and compartment-specific regulation of acidification.