A 30-amino-acid peptide corresponding to variable domain IV (VD IV) of the major outer membrane protein of Chlamydia trachomatis serovar E was conjugated to keyhole limpet hemocyanin (KLH) and used to immunize mice. The resulting antisera (anti-KLH-VD IV sera) recognized all 15 serovars of C. trachomatis when assayed by indirect immunofluorescence and Western blotting. Probing of overlapping hexameric peptides representing VD IV with mouse anti-KLH-VD IV sera revealed that two main regions of the peptide were recognized by the antisera, the N terminus of the peptide, which contains B-complex-specific epitopes, and the middle region of the peptide, which contains a species-conserved domain. When used in an in vitro neutralization assay, these antisera were able to neutralize mainly serovars in the B complex. These data provide evidence that a linear peptide corresponding to VD IV can induce in vitro protection from C. trachomatis infectivity that is subspecies specific.