Ehrlichia ewingii, a tick-transmitted rickettsia previously known only as a canine pathogen, was recently recognized as a human pathogen. E. ewingii has yet to be cultivated, and there is no serologic test available to diagnose E. ewingii infection. Previously, a fragment (505 bp) of a single E. ewingii gene homologous to 1 of 22 genes encoding Ehrlichia chaffeensis immunodominant major outer membrane proteins 1 (OMP-1s)/P28s was identified. The purposes of the present study were to (i) determine the E. ewingii omp-1 gene family, (ii) determine each OMP-1-specific peptide, and (iii) analyze all OMP-1 synthesized peptides for antigenicity. Using nested touchdown PCR and a primer walking strategy, we found 19 omp-1 paralogs in E. ewingii. These genes are arranged in tandem downstream of tr1 and upstream of secA in a 24-kb genomic region. Predicted molecular masses of the 19 mature E. ewingii OMP-1s range from 25.1 to 31.3 kDa, with isoelectric points of 5.03 to 9.80. Based on comparative sequence analyses among OMP-1s from E. ewingii and three other Ehrlichia spp., each E. ewingii OMP-1 oligopeptide that was predicted to be antigenic, bacterial surface exposed, unique in comparison to the other E. ewingii OMP-1s, and distinct from those of other Ehrlichia spp. was synthesized for use in an enzyme-linked immunosorbent assay. Plasmas from experimentally E. ewingii-infected dogs reacted significantly with most of the OMP-1-specific peptides, indicating that multiple OMP-1s were expressed and immunogenic in infected dogs. The results support the utility of the tailored OMP-1 peptides as E. ewingii serologic test antigens.