A new Ehrlichia strain, designated as Ehrlichia chaffeensis, Sapulpa strain, was isolated from a patient from Oklahoma with severe ehrlichiosis. Isolation of the ehrlichial pathogen was achieved by inoculating patient blood onto HEL cells and DH82 cells. Antigenic properties of the new isolate were characterized with monoclonal antibodies, homologous patient serum, and polyclonal rabbit serum by Western immunoblotting. The results showed antigenic differences and protein size variation of Sapulpa strain compared with the other 2 strains of E. chaffeensis. Sequencing of the 16S rRNA gene showed 100% identity to that of E. chaffeensis, strain 91HE17. Polymerase chain reaction and sequencing of DNA homologous to the 120-kDa protein gene of E. chaffeensis, Arkansas strain, showed that this gene of Sapulpa strain was smaller than that of Arkansas strain and contained a repeat region with three tandem repeat units.