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Publisher Summary Most studies on the intracellular behavior of mycobacteria are based on observations using host cells from mice or humans that are infected with a variety of mycobacterial species. Mouse peritoneal macrophages or human monocytes or monocyte-derived macrophages have generally been used in the case of Mycobacterium tuberculosis or Bacillus Calmette–Guerin (BCG). The use of cell lines for long term studies on the survival of mycobaeteria is, however, hindered by the cell growth rate, which is more rapid than that of most pathogenic mycobacteria. Artificial reduction of the cell growth rate should be avoided as it may give rise to misleading conclusions to the extent that cells may be affected in fundamental functions, including endocytosis and microbicidal activity. This chapter discusses the endocytic pathway, phagosomes, and mycobacteria. In many naturally occurring instances, mycobacteria may be phagocytosed in an opsonized state or as bacterial clumps, and this may lead to their delivery into phagolysosomes (see above). The specific interactions of mycobacteria with early endosome-derived membrane constituents may provide a rescue mechanism if mycobacteria can temporarily survive in phagolysosomes.