Prospective surveillance of 63 human immunodeficiency virus (HIV)-positive patients and 9 HIV-negative partners over 5-27 months yielded 51 adenoviruses from 18 HIV-positive patients. These were serotyped and compared by restriction enzyme analysis (REA) together with 24 isolates from 19 other HIV-positive patients. The actuarial risk of infection at 1 year in HIV-positive patients was 28% (17% with entry CD4 cell count of > 200/mm3 and 38% with CD4 cell count of < or = 200/mm3, P = .03). The most frequent site of infection was gastrointestinal (17/18 patients) with mainly subgenus D adenoviruses, while urinary infection was caused by subgenus B or D. Prolonged fecal excretion (2-27 months) was associated with CD4 cell counts < 150/mm3. Identical strains were seen in 2 HIV-positive partners and 2 unrelated patients. Gastrointestinal infection was temporally associated with diarrhea in only 7 (41%) of 17 cases. The remainder (59%) were asymptomatic or minimally symptomatic, and diarrhea was often caused by other opportunistic pathogens.