When Chlamydia trachomatis elementary bodies enter epithelial cells, they occupy membrane-bound vesicles that aggregate with each other in a calcium-dependent manner but that do not fuse with lysosomes. As members of the annexin family of calcium- and membrane-binding proteins have been implicated in mediating calcium-regulated membrane traffic during endo- and exocytosis, we examined the intracellular localization of certain annexins following invasion of HeLa and McCoy cells by C. trachomatis serovar L2. Immunofluorescence staining with a panel of polyclonal antibodies against five human annexins revealed that annexins III, IV, and V translocate within the cytoplasm to the proximity of intracellular chlamydiae whereas the distribution of annexins I and VI was unaffected. The distinct distribution of annexins I and III was further analyzed by confocal microscopy, which revealed an intimate association between chlamydial aggregates or inclusions and annexin III. Confocal microscopy also confirmed the nonassociation of annexin I with chlamydial aggregates. Depletion of intracellular Ca2+ did not prevent association of annexin III with individual elementary body-containing endosomes but did prevent formation of chlamydial aggregates and translocation of annexin III. Furthermore, chloramphenicol-treated cells also showed association between chlamydial aggregates and annexin III, indicating that the annexins are of host cell origin. These data suggest that certain cytosolic annexins may be involved in the Ca(2+)-dependent aggregation and fusion of chlamydia-containing vesicles. The fact that these Ca(2+)-binding proteins differ in their ability to associate with chlamydia-containing vesicles and inclusions implies that the factors that regulate the interaction of annexin I and annexin III with membrane are different and suggests a selective regulatory mechanism for endosome aggregation and avoiding lysosome fusion during chlamydia infection.