Adenoviruses (AdV) cause diseases that range from localized, self-limited illnesses to fatal infections in immunocompromised patients. Culture is assumed to be sensitive but requires viable virus and up to 3 weeks for detection, and it can be inhibited by bacterial contamination. A new PCR method amplifying a region of the hexon gene was developed in order to detect AdV in urine more rapidly and with greater sensitivity than obtainable by culture technology. All 18 serotypes tested were detected. Quantitatively, with optimized urine processing, AdV PCR detected 0.2 PFU/ml (serotype 11) and 10 DNA copies/ml (serotype 2). Serially collected urine samples from human immunodeficiency virus (HIV)-infected patients with concurrent cytomegalovirus retinitis were divided into three groups: AdV culture-positive samples, AdV culture-negative or bacterially contaminated samples from patients with a history of AdV culture-positive urines, and AdV culture-negative samples from patients without a history of AdV culture positivity. Urine samples from healthy adults were also tested by culture and PCR to screen for asymptomatic shedding. Amplification was assessed with and without prior DNA purification. AdV was detected by PCR in 90% of culture-positive urines (100% of unclotted samples, e.g., those culture positive after storage for PCR testing), 71% of culture-negative or bacterially contaminated urines from AdV-infected patients, and 28% from AdV culture-negative patients. Healthy volunteers were culture negative for AdV, and 96% were PCR negative. The new AdV PCR method is rapid and sensitive and can detect viral DNA in samples for which culturing is problematic. The role of AdV replication during HIV infection merits further investigation with sensitive tools such as PCR.