Endemic (type 1, 2, 5 and 6) and epidemic (type 3, 4 and 7) respiratory adenovirus infections are associated with upper respiratory tract symptoms, pharyngoconjunctival fever, and pneumonia. Improved methods of diagnosis are needed, particularly in immunocompromized patients. We examined 93 throat swabs or nasopharyngeal aspirates from patients with acute respiratory disease using virus isolation and an adenovirus-specific polymerase chain reaction (PCR) based on consensus primers H1 and H2 derived from the hexon region DNA sequences of serotypes 2 and 5. Specimens which yielded viruses other than adenovirus in cell culture (n = 23) or which were negative for infectious viruses (n = 25) were negative in the PCR. The sensitivity of DNA amplification was 76% (34/45) in comparison with virus culture, being markedly lower with subgenus B (types 3 and 7) strains than with subgenus C (type 1, 2, 5 and 6) isolates (8/16 (50%)) vs. 26/28 (93%). P = 0.004) despite the use of a low annealing temperature to maximize detection of adenoviruses belonging to subgenera other than C. Of the 11 samples falsely negative in a single-round PCR but yielding adenovirus type 1 (n = 1), type 2 (n = 1). type 3 (n = 7), type 7 (n = 1), or untyped isolates (n = 1) in cell culture, nine (82%) gave positive results after nested DNA amplification. Possible approaches to further improving the performance of adenovirus PCR with respiratory specimens are discussed.