This review focuses on events involved in the biogenesis of the lysosome. This organelle contains a diverse array of soluble, luminal proteins capable of digesting all the macromolecules in the cell. Altered function of lysosomes or its constituent enzymes has been implicated in a host of human pathologies, including storage diseases, cancer, and infectious and neurodegenerative diseases. Luminal enzymes are well-characterized, and aspects of how they are incorporated into lysosomes are known. However, little is known about the composition of the membrane surrounding the organelle or how the membrane is assembled. Our starting point to study lysosome biogenesis is to define the composition of the membrane by the use of proven methods for purification of lysosomes to near homogeneity and then to characterize membrane-associated and integral lysosomal membrane proteins. This has been achieved using advanced proteomics (electrophoretic or chromatographic separations of proteins followed by time-of-flight mass spectrometric identification of peptide sequences). To date, we have identified 55 proteins in the membrane-associated fraction and 215 proteins in the integral membrane. By applying these methods to mouse models of lysosome dysgenesis (such as BEIGE, Pale Ear, PEARL) that are related to human diseases such as Chediak-Higashi and Hermansky-Pudlak syndromes, it may be possible to define the membrane protein composition of lysosomes in each of these mutants and to determine how they differ from normal. Identifying proteins affected in the respective mutants may provide hints about how they are targeted to the lysosomal membrane and how failure to target them leads to disease; these features are pivotal to understanding lysosome biogenesis and have the potential to implicate lysosomes in a broad range of human pathologies.