Twenty-seven monoclonal antibodies (mAb), eight with subclass-specificities and nineteen reacting with one or two C. trachomatis serovars, were developed and used to immunotype twenty-six clinical isolates from Greek patients with chlamydial infections. Twelve samples, first classified as serovar D, were identified as the recently established serovar Da on the basis of their negative reaction with mAb JG9, an mAb previously shown to distinguish between D and Da, and a new mAb 114D9 with similar specificity. The 12 Da strains represent the highest prevalence of the rare serovar Da that has been reported worldwide. Further characterization of mAbs JG9 and 114D9 revealed a unique cross-reactivity of the D-specific JG9 with the mouse biovar MoPn. Epitope mapping studies on overlapping synthetic peptides of the fourth variable domain of the MOMP of serovar D and Da localized the epitopes of JG9 and 114D9 and a D/Da specific mAb 113D5 within this domain. The results were consistent with the specificity of these mAbs observed with whole microorganisms and indicated their usefulness as epidemiologic markers.