Genetic and technical advances of the past few years have allowed us to test some of the vacuolar trafficking and vacuole biogenesis models that had been previously proposed mainly on the basis of morphological and immunolocalization studies. We have now tools to start answering some fundamental questions such as: How are vacuoles formed? Are all vacuoles formed similarly? Do different types of vacuoles coexist in a cell? How are proteins sorted to the vacuole? How many trafficking pathways to vacuoles exist? Can there be trafficking to two types of vacuoles simultaneously? Last but not least, how do vacuoles balance the continuous flow of new materials, cargo and membrane, and maintain their volume? We will review recent data trying to answer these questions and propose some models that accommodate the results obtained.