The phagocytic function of neutrophils is a crucial element in the host defence against invading microorganisms. We investigated phagocytosis and intracellular killing of an attenuated strain of Mycobacterium tuberculosis(H37Ra) by human neutrophils focusing on the role of the cytosolic free calcium concentration [Ca2+]i and certain cytosolic calcium-dependent membrane-binding proteins annexins. Phagocytic uptake did not trigger a calcium rise and occurred independently of different calcium conditions, and in a serum-dependent manner. Changes in the viability of H37Ra were determined by agar plate colony count and a radiometric assay. Neutrophils showed a capacity to kill ingested mycobacteria and this occurred without a rise in [Ca2+]i. The ability to kill H37Ra decreased in the absence of extracellular calcium and when intra-extracellular calcium was reduced. Immunofluorescence staining revealed that during phagocytosis of H37Ra, annexins III, IV and VI translocated from cytoplasm to the proximity of the H37Ra-containing phagosomes, whereas the localization of annexin I and V remained unchanged. The translocation of annexin IV occurred even when Ca2+-depleted neutrophils ingested H37Ra in the absence of extracellular calcium. We concluded that neutrophil-mediated killing of mycobacteria is a Ca2+-dependent process. The fact that the association of certain annexins to the membrane vesicle containing H37Ra differ from other phagosomes suggests a selective regulatory mechanism during phagocytosis of mycobacteria by neutrophils.