Phagocytic antigen-presenting cells (APCs) are involved in innate and adaptive immune responses to bacteria. Adaptive responses to bacteria involve processing of bacterial antigens for presentation by class II major histocompatibility complex (MHC II) molecules and class I MHC (MHC I) molecules to stimulate CD4(+) and CD8(+) T cells, respectively. To examine the role of phagosomes in processing of antigens for presentation by MHC II molecules to CD4(+) T cells, phagosomes have been biochemically and functionally analyzed by a variety of techniques that include flow analysis (flow organellometry), SDS-PAGE/Western blotting, and an antigen-presenting organelle assay. Using these techniques, we have demonstrated that phagosomes containing latex beads or Mycobacterium tuberculosis (MTB) contain components of the MHC II processing pathway and support the formation of peptide-MHC II complexes.