A gene encoding a 28-kDa protein of Ehrlichia canis was cloned, sequenced, and expressed, and a comparative molecular analysis with homologous genes of E. canis, Cowdria ruminantium, and Ehrlichia chaffeensis was performed. The complete gene has an 834-bp open reading frame encoding a protein of 278 amino acids with a predicted molecular mass of 30.5 kDa. An N-terminal signal sequence was identified, suggesting that the protein undergoes posttranslational modification to a mature 27.7-kDa protein (P28). The E. canis p28 gene has significant nucleic acid and amino acid sequence homologies with the E. chaffeensis outer membrane protein-1 (omp-1) gene family, with the Cowdria ruminantium map-1 gene, and with other E. canis 28-kDa-protein genes. Southern blotting revealed the presence of at least two additional homologous p28 gene copies in the E. canis genome, confirming that p28 is a member of a polymorphic multiple-gene family. Amino acid sequence analysis revealed that E. canis P28 has four variable regions, and it shares similar surface-exposed regions, antigenicity, and T-cell motifs with E. chaffeensis P28. The p28 genes from seven different E. canis isolates were identical, indicating that the gene for this major immunoreactive protein is highly conserved. In addition, reactivity of sera from clinical cases of canine ehrlichiosis with the recombinant P28 demonstrated that the recombinant protein may be a reliable serodiagnostic antigen.