The chlamydial cryptic plasmid encodes eight putative open reading frames (ORFs), designated pORF1 to -8. Antibodies raised against these ORF proteins were used to localize the endogenous proteins during chlamydial infection. We found that the pORF5 protein (also known as pgp3) was detected mainly in the cytosol of Chlamydiainfected cells, while the remaining seven proteins were found inside the chlamydial inclusions only. The pgp3 distribution pattern in the host cell cytosol is similar to but not overlapping with that of chlamydial protease/ proteasome-like activity factor (CPAF), a chlamydial genome-encoded protein known to be secreted from chlamydial inclusions into the host cell cytosol. The anti-pgp3 labeling was removed by preabsorption with pgp3 but not CPAF fusion proteins and vice versa, demonstrating that pgp3 is a unique secretion protein. This conclusion is further supported by the observation that pgp3 was highly enriched in cytosolic fractions and had a minimal presence in the inclusion-containing nuclear fractions prepared from Chlamydia-infected cells. The pgp3 protein was detected as early as 12 h after infection and was secreted by all chlamydial species that carry the cryptic plasmid, suggesting that there is a selection pressure for maintaining pgp3 secretion during chlamydial infection. Although expression of pgp3 in the host cell cytosol via a transgene did not alter the susceptibility of the transfected cells to the subsequent chlamydial infection, purified pgp3 protein stimulated macrophages to release inflammatory cytokines, suggesting that pgp3 may contribute to chlamydial pathogenesis.Chlamydia represents a group of obligate intracellular bacterial pathogens consisting of many different species and causing various health problems in both humans and animals. The species C. trachomatis is composed of more than 15 different serovars (including A to L serovars); some infect the human ocular epithelium, potentially leading to preventable blindness (64), while others infect human urogenital tract tissue, which can potentially cause severe complications such as ectopic pregnancy and infertility (56). C. muridarum, formerly known as the murine biovar of C. trachomatis (designated MoPn, with the single strain Nigg), has been extensively used to study C. trachomatis pathogenesis and immunology in mouse models (32,37,38,42,43). Although most isolates of C. pneumoniae organisms often infect the human respiratory system, causing respiratory pathologies and exacerbating lesions in the vascular wall (3, 28), the C. pneumoniae N16 strain has been isolated from equines (60). Other chlamydial species that mainly infect animals include C. caviae (50), C. psittaci (47), C. abortus (66), and C. felis (1). Despite the apparent differences in tissue tropism, all chlamydial species share similar genome sequences (1,29,49,50,66) and possess a common intracellular growth cycle with distinct biphasic stages (24). The chlamydial intracellular infection-induced inflammation is considered a major cause of Chlamydi...