To localize and characterize the hypothetical protein CT358 in the chlamydial infected cells. CT358 gene from the Chlamydia trachomatis(C. trachomatis) serovar D genome was amplified and cloned into the pGEX and pDSRedC1 vectors. The recombinant plasmid pGEX-CT358 was constructed and expressed as GST fusion proteins. The GST-CT358 fusion protein was used to immunize mice to raise the antibodies, which specifically recognized CT358 without cross-reacting with other unrelated proteins. The antibodies were then used to localize the endogenous CT358 protein and determine the expression pattern in Chlamydial infected cells using an indirect immunofluorescence assay (IFA). Meanwhile, pDSRedC1-CT358 recombinant plasmid was transfected to HeLa cells to evaluate the effect of CT358 expression on the subsequent chlamydial infection. The hypothetical protein CT358 was identified in the inclusion membrane of C. trachomatis-infected cells for the first time,and it was detected as early as 12 h after C. trachomatis infection and remained in the inclusion membrane throughout the rest of the infection cycle. Cytosolic expression of CT358 via a transgene failed to affect the subsequent chlamydial infection. These observations together have demonstrated that CT358 is a newly identified chlamydial inclusion membrane protein, giving the potentially importance for further understanding the mechanisms of chlamydial intracellular parasitism.