Whipple disease is a rare infection characterized clinically by diarrhea, fever, weight loss, arthralgia, malabsorption, and other systemic manifestations. The etiologic agent, Tropheryma whipplei, has been cultured only rarely. By using a polyclonal rabbit antibody produced against a cultured strain of T whipplei, tissue sections from 18 patients with Whipple disease were studied. Specimens from patients with histologic mimics and other infections served as control specimens. Immunostaining was identified in all 18 patients. Granular immunostaining was observed similar to that in periodic acid-Schiff (PAS) stains. In 2 patients, immunostaining was identified in specimens negative by H&E and PAS stains. In 4 patients studied before and after antibiotic therapy, immunostaining was retained but diminished in intensity and quantity. Immunostaining was not identified in any control specimen. Immunohistochemical analysis is a sensitive and specific method for the diagnosis of Whipple disease in paraffin-embedded tissue and may provide new opportunities to investigate the pathogenesis of the infection.