Three monoclonal antibodies (MAbs), E4, Ll-4, and L1-24, to the major outer membrane protein (MOMP) of Chiamydia trachomatis were identified that neutralized in vitro the infectivity of members of the Band C-related complex as well as the mouse pneumonitis strain. MAbs L1-4, Ll-24, and E4 gave a strong signal in an indirect immunofluorescence assay and/or Western immunoblot with all serovars of the lymphogranuloma venereum and trachoma biovars and a weak signal with the mouse biovar. In addition, C. psittaci and C. pneumoniae were also weakly recognized by MAbs Ll-4 and Ll-24. As determined by the technique of overlapping peptides, all three MAbs showed reactivity to variable domain (VD) IV of MOMP. While all three MAbs had different recognition patterns, all strongly bound to the peptides TLNPTI and LNPTIA within the species-conserved region of VD IV. MAb E4 also recognized the peptide SATAIF in the subspecies region of VD IV. Peptides corresponding to VY IV of MOMP were synthesized and used in competitive inhibition experiments to determine the functional location of the epitope recognized by these three MAbs. Both the serological and neutralizing activities of MAb E4 were inhibited by the peptides ATAIFDTTTLNPTIAG and FDTTTLNPTIAG; however, none of the peptides made to the VD IV region blocked the neutralizing activity of MAbs L1-4 and Ll-24. Therefore, the neutralizable domain of the epitope recognized by MAb E4 is contiguous and may be an important candidate for inclusion in a subunit vaccine.