Objective To determine the prevalence of HIV in endoscopic biopsies from the esophagus, stomach, duodenum, and rectum of homosexual and bisexual men at various stages of HIV infection as part of a comprehensive study of gastrointestinal dysfunction in HIV infection. Methods After repeated washings and mechanical disruption, biopsies obtained from 58 volunteers were individually cocultured with pooled peripheral blood lymphocytes from healthy HIV-seronegative blood donors. Results HIV was isolated from at least one site in 40 out of 49 patients. Esophageal biopsies were most frequently found positive (46%), followed by duodenal biopsies (44%), rectal biopsies (43%), and gastric biopsies (27%). Recovery of HIV was not related to any gastrointestinal signs or symptoms. HIV was recovered in the biopsies both from asymptomatic patients with CD4 lymphocyte counts > 500 × 106/l and also from patients with more advanced disease receiving zidovudine therapy. Conclusions The entire gastrointestinal tract appears to be a target site throughout the course of HIV infection in homosexual and bisexual men.