The aim of this study was to evaluate the applicability of diagnostic methods for dual-infected cases of human adenoviruses (AdVs) and coxsackieviruses type B (CBs). For this purpose, 100 nasopharyngeal samples from patients with acute exudative tonsillitis and clinically suspected AdV infection were analyzed. Using PCR and real-time PCR techniques for AdVs and CBs, we found 86 AdVs-only positive samples; we also found five dual-infected samples containing 5.4 x 10(5) to 7.1 x 10(8) copies/mL of AdV genomes and 1.4x104 to 1.3 x 10(9) copies/mL of CB genomes. By viral culture using A549 cells, two co-infected samples, which contained over 10(8) copies/mL of AdV genomes and <10(5) copies/mL of CB genomes, became AdV dominant, while three samples with less than 2.0 x 10(6) copies/mL of AdV genomes became CB dominant. An immunochromatography kit for diagnosing AdVs at the bedside was positive for 3/5 dual-infected patients, and PCR techniques for AdVs and CBs were both positive for 5/5. Viral culture is usually considered to be the gold standard for AdV diagnosis, but our results demonstrate the importance of PCR applications for the detection of AdV and CB genomes, particularly in clinical cases of suspected AdV infection. Even though the sample size of dual infection (n=5) is small, our results show the existence of dual infection cases which were difficult to diagnose by viral culture alone.