Disease-causing microbes utilize various strategies to modify their environment in order to create a favorable location for growth and survival. Gram-negative bacterial pathogens often use specialized secretion systems to translocate effector proteins directly into the cytosol of the eukaryotic cells they infect. These bacterial proteins are responsible for modulating eukaryotic cell functions. Identification of the bacterial effectors has been a critical step toward understanding the molecular basis for the pathogenesis of the bacteria that use them. Chlamydiae are obligate intracellular bacterial pathogens that have a type III secretion system believed to translocate virulence effector proteins into the cytosol of their host cells. Selective permeabilization of the eukaryotic cell membrane was used in conjunction with metabolic labeling of bacterial proteins to identify chlamydial proteins that localize within the cytosol of infected cells. More than 20 Chlamydia trachomatis and C. pneumoniae proteins were detected within the cytoplasmic compartment of infected cells. While a number of cytosolic proteins were shared, others were unique to each species, suggesting that variation among cytosolic chlamydial proteins contributes to the differences in the pathogenesis of the chlamydial species. The spectrum of chlamydial proteins exported differed concomitant with the progress of the developmental cycle. These data confirm that a dynamic relationship exists between Chlamydia and its host and that translocation of bacterial proteins into the cytosol is developmentally dependent.