Sera from 35 patients diagnosed with human granulocytic ehrlichiosis in Connecticut were tested by indirect IFA staining methods with 5 strains of Ehrlichia equi or the human granulocytic ehrlichiosis agent to assess the suitability of different strains in laboratory analyses. Antigens included horse-derived infected neutrophils (MRK and BDS strains) and human isolates cultured in human promyelocytic leukemia cells (NCH-1, RCH, and Webster). Of 35 sera, 23 (65.7%) reacted to all 5 strains. Seropositivity was highest (97.1%) in assays that contained the MRK strain from California and lowest (71. 4%) in tests with the NCH-1 strain from Nantucket, Massachusetts. In parallel testing of 32 sera with the NCH-1 strain by indirect IFA and Western blot analyses, results were concordant for 30 samples (93.8%). All strains of ehrlichiae can be used in IFA analyses for antibody detection, but assay sensitivity varied with the strain used.