Pathogenic mycobacteria infect macrophages where they replicate in phagosomes that minimize contact with late endosomal/lysosomal compartments. Loading of Ags to MHC class II molecules occurs in specialized compartments with late endosomal characteristics. This points to a sequestration of mycobacteria-containing phagosomes from the sites where Ags meet MHC class II molecules. Indeed, in resting macrophages MHC class II levels decreased strongly in phagosomes containing M. avium during a 4-day infection. Phagosomal MHC class II of early (4 h) infections was partly surface-derived and associated with peptide. Activation of host macrophages led to the appearance of H2-M, a chaperon of Ag loading, and to a strong increase in MHC class II molecules in phagosomes of acute (1 day) infections. Comparison with the kinetics of MHC class II acquisition by IgG-coated bead-containing phagosomes suggests that the arrest in phagosome maturation by mycobacteria limits the intersection of mycobacteria-containing phagosomes with the intracellular trafficking pathways of Ag-presenting molecules.