Ehrlichia chaffeensis and E. canis have a small subset of tandem repeat (TR)-containing protein orthologs, including p120/p140, which elicit strong antibody responses. The TR regions of these protein orthologs are immunoreactive, but the molecular characteristics of the p120/p140 epitopes have not been determined. In this study, the immunodeterminants of the E. chaffeensis p120 and E. canis p140 were identified and molecularly defined. Major antibody epitope-containing regions of both p120 and p140 were localized to the TR regions, which reacted strongly by Western immunoblotting with antibodies in sera from E. chaffeensis-infected dogs or patients and E. canis-infected dogs, respectively. Single continuous species-specific major epitopes within the E. chaffeensis p120 and E. canis p140 TRs were mapped to homologous surface-exposed glutamate/aspartate-rich regions (19 to 22 amino acids). In addition, minor cross-reactive epitopes were localized to homologous N- and C-terminal regions of p120 and p140. Furthermore, although the native and recombinant p120 and p140 proteins exhibited higher-than-predicted molecular masses, posttranslational modifications were not present on abnormally migrating p120 and p140 TR recombinant proteins as determined by matrix-assisted laser desorption ionization-time of flight mass spectrometry.