Acidification of some organelles, including the Golgi complex, lysosomes, secretory granules, and synaptic vesicles, is important for many of their biochemical functions. In addition, acidic pH in some compartments is also required for the efficient sorting and trafficking of proteins and lipids along the biosynthetic and endocytic pathways. Despite considerable study, however, our understanding of how pH modulates membrane traffic remains limited. In large part, this is due to the diversity of methods to perturb and monitor pH, as well as to the difficulties in isolating individual transport steps within the complex pathways of membrane traffic. This review summarizes old and recent evidence for the role of acidification at various steps of biosynthetic and endocytic transport in mammalian cells. We describe the mechanisms by which organelle pH is regulated and maintained, as well as how organelle pH is monitored and quantitated. General principles that emerge from these studies as well as future directions of interest are discussed.