C5a is a powerful proinflammatory and immunomodulatory mediator as evidenced by its involvement in infectious, allergic, and autoimmune diseases as well as in cancer. C5a exerts most of its biologic functions through binding and activation of the G protein-coupled C5a receptor (C5aR). C5aR activation promotes complex signaling pathways eventually resulting in increased intracellular calcium (Ca(2+))i concentration. One of the prototypic effector functions associated with C5aR activation is the reconfiguration of the cell cytoskeleton resulting in increased cell motility and ligand-specific cell migration. Here, we describe three assays to assess C5a-mediated effector functions: (1) increase in (Ca(2+))i in bone marrow-derived neutrophils using the intracellular fluorescent Ca(2+)-sensitive indicator Fluo-4 AM and flow cytometry-based methods; (2) in vitro migration of murine, bone marrow-derived neutrophils using a modified Boyden chamber; and (3) in vivo migration of neutrophils from the circulation into the peritoneal cavity. To control for the specificity of the C5a-mediated effects, C5aR-deficient mice, neutralizing C5aR-specific antibodies and potent pharmacologic C5aR inhibitors are available.