Microorganisms have long been suspected of causing Crohn's disease (CD); however, an etiologic agent has yet to be identified. Few studies have employed immunocytochemistry (ICC) to examine tissue from patients with CD for microbial antigens. We investigated 36 formalin-fixed tissues from 16 patients with CD with ICC. No evidence of adenovirus, Borrelia, Brucella, BVDV, Campylobacter, Campylobacter-like organisms, Chlamydia, coronavirus, CMV, EBV, Legionella, mycobacteria, Pseudomonas, rotavirus, Salmonella, Shigella, staphylococci, Toxoplasma gondii, Treponema, or Yersinia was found. ICC identified E. coli and streptococcal antigens in 11 (69%) and 10 (63%) of the 16 cases studied, respectively. Escherichia coli immunoreactivity was located in ulcers, within the lamina propria, and along fissures. Streptococcal immunolabeling occurred within mucosal epithelial cells, in the lamina propria, in ulcers, along fissures, in granulomatous inflammation including multinucleate giant cells, and in lymph nodes. These results suggest that some of the granulomas in CD may result from immunologic processing of bacterial antigens following their penetration through a compromised mucosa. E. coli and streptococcal antigens may contribute to the pathogenesis of CD.