Ehrlichia chaffeensis, the novel etiologic agent of human ehrlichiosis in the United States, was first isolated in 1990 and reported in 1991. To analyze the antigenic components of E. chaffeensis, we cultivated these obligate intracellular bacteria in DH82 cells, purified the ehrlichiae by renografin density gradient centrifugation, and examined the antigens by Western immunoblotting. Rabbit and human antisera to E. chaffeensis revealed more than 20 bands ranging from 20 to 200 kD. The distinct 22-kD protein was heat labile. The rest of the major immunoreactive components were heat stable. The immunoblots of E. chaffeensis were highly similar when probed with antisera to E. chaffeensis, E. canis, and E. ewingii, indicating the close antigenic relationships among the three species. The 22-kD protein cross-reacted only with anti-E. canis serum. The antibody against E. sennetsu reacted strongly with the 66-, 64-, 55-, and 44-kD antigens of E. chaffeensis. The E. risticii antisera reacted strongly with the 55- and 44-kD bands but only faintly with the 66-kD band. The major immunoreactive antigens of E. chaffeensis (66, 55, and 44 kD) showed cross-reactions with all the different antisera tested. The results indicated that E. chaffeensis is antigenically most closely related to E. canis, is less closely related to E. ewingii, and is only distantly related to E. sennetsu and E. risticii.