Plasmid pCT is present in essentially all isolates of Chlamydia trachomatis and may encode factors important for survival in the natural environment. However, no pCT-associated phenotype has been described so far. With the purpose of investigating the possibility of a role of pCT in C. trachomatis pathogenicity we examined the expression of an ORF (ORF3), potentially encoding a 28 kDa polypeptide (pgp3). Analysis of RNA extracted from chlamydia-infected Vero cells detected ORF3-specific transcripts, from 20 h post-infection onwards, mainly as discrete RNA species of 1390 nucleotides comprising the downstream ORF4 sequence. ORF3 DNA was cloned and expressed in Escherichia coli as a 39 kDa fusion protein (MS2/pgp3). Antibodies raised against purified MS2/pgp3, specifically recognized a 28 kDa protein on Western blots of protein from purified chlamydial elementary bodies (EBs). The same antibodies detected chlamydial inclusions in methanol-fixed infected cells by immunofluorescence. Western blot analysis of EBs extracted with 2% Sarkosyl, showed that a large proportion of the 28 kDa antigen is associated with the detergent-insoluble ('membrane') fraction. Antibodies recognizing pgp3 epitopes were detected in sera from patients with chlamydial infections, but not in sero-negative control sera. The finding support the hypothesis that pCT may provide a function related to chlamydial cell physiology.