Peptides representing the Chlamydia trachomatis major outer membrane protein variable domains (VD) 1 and 4 of serovars C and E, respectively, have been shown to elicit a neutralizing antibody response in mice. To assess whether the position within a chimeric peptide influences the immunogenicity of the epitopes, two constructs, VD 1-4 and VD 4-1, were made in which the position of the VD relative to the amino and carboxy terminals were rotated. C57BL/10 mice were immunized with 100 micrograms of peptide in complete Freund's adjuvant (FA) on day 0, followed by an immunization with peptide (100 micrograms) in complete FA on day 14. By day 21 the immunodominant epitope in both chimeras as measured by ELISA was the one located at the carboxy terminus. A pepscan of the VD 1-4 antisera revealed a main peak in VD 4 which had been previously identified by neutralizing MAbs. The VD 4-1 antisera gave a peak in the VD 1 region which did not correspond to regions previously mapped with neutralizing MAbs. The VD 1-4 antisera but not the VD 4-1 antisera was able to neutralize in vitro serovar E. In summary, the position of these chlamydial epitopes within a chimeric peptide greatly influenced the resulting immune response.