Antigenic determinants were identified from seven chlamydial major outer membrane proteins by using overlapping hexapeptides and polyclonal antisera. Sixty-one determinants were detected, and 30 were surface exposed on the native organisms. The two negatively charged residues, aspartic acid and glutamic acid, were found most often in determinants. Thirteen antigenic sites were further characterized by alanine substitution. Differences in fine specificities of these linear determinants were observed in alanine substitution profiles. Five determinants had adjacent critical residues, while eight had critical residues alternated with noncritical residues. Complete replacement analysis of two antigenic determinants provided more detailed information for elucidating the structural basis of the specificity of antigen-antibody interaction and suggested a correlation between sequence conservation and tolerance to amino acid substitution for antigenic sites subject to intense immune selection pressure.