Standard immunoperoxidase techniques were evaluated in the diagnosis of Rocky Mountain spotted fever (RMSF). Formalin-fixed, paraffin-embedded tissue was tested to detect Rickettsia rickettsii using the same antibody provided by the Centers for Disease Control and Prevention that is used for direct immunofluorescence (DIF). Tissues from 23 patients with suspected RMSF were divided: some were snap-frozen for DIF; the remainder were fixed in formalin, processed routinely, and embedded in paraffin for immunoperoxidase and hematoxylin and eosin staining. Ten patients were ultimately determined to have RMSF; in nine of these patients, both DIF and immunoperoxidase staining were positive for R rickettsii. There were no discrepancies, positive or negative, between the two methods. In the one case with a false-negative result by both methods, the patient had received antirickettsial antibiotics 72 hours before the biopsy was performed. These data demonstrate that the immunoperoxidase technique is effective in diagnosing RMSF and may be applied to cases retrospectively.