Chlamydia trachomatis is a bacterial obligate intracellular parasite that replicates within a vacuole, termed an inclusion, that does not fuse with lysosomes. Within 2 h after internalization, the C. trachomatis inclusion ceases to interact with the endocytic pathway and, instead, becomes fusogenic with exocytic vesicles containing exogenously synthesized NBD-sphingomyelin. Both fusion of exocytic vesicles and long-term avoidance of lysosomal fusion require early chlamydial gene expression. Modification of the chlamydial inclusion probably occurs through the expression and insertion of chlamydial protein(s) into the inclusion membrane. To identify candidate inclusion membrane proteins, antisera were raised against a total membrane fraction purified from C. trachomatis-infected HeLa cells. By indirect immunofluorescence, this antisera recognized the inclusion membrane and, by immunoblot analysis, recognized three chlamydial-specific antigens of approximate molecular weights 15, 18 and 21 kDa. IncG, encoding an 18 kDa and 21 kDa doublet chlamydial antigen, was identified by screening a C. trachomatis, serovar L2, genomic expression library. Three additional genes, incD, incE and incF, were co-transcribed with incG. Monospecific antisera against each of the four genes of this operon demonstrated that the gene products were localized to the chlamydial inclusion membrane. Immediately downstream from the operon containing incD-G was the C. trachomatis homologue of incA. Like IncD, E, F and G, C. trachomatis IncA is also localized to the inclusion membrane. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis demonstrated that IncD-G, but not incA, are transcribed within the first 2 h after internalization, making them candidates for chlamydial factors required for the modification of the nascent chlamydial inclusion.