A total of 189 conjunctival scrapings were collected from patients in Tokyo, Japan by monitoring adenovirus infection in community-based clinics during 2005 and 2006. Of the 189 samples, 155 (82%) had adenoviruses detected by polymerase chain reaction (PCR). The serotypes were determined by PCR-restriction fragment length polymorphism (PCR-RFLP) analysis, using a combination of endonucleases, such as HhaI, AluI, and HaeIII, and neutralization tests (NTs). PCR-RFLP identified five serotypes: serotype 3: 16.8%, serotype 4: 9.7%, serotype 8: 34.8%, serotype 11: 23.2%, and serotype 37: 15.5%. Adenovirus 8 was the most common serotype identified. A subset consisting of 25 isolates identified as adenovirus 8 from this study plus 25 isolates from Kawasaki were analyzed using PCR sequencing of the hexon gene. Compared with prototype adenovirus serotype 8 and serotype 9 derived in Tokyo and Kawasaki, these isolates shared 61.7-62.8% and 80.5-82.7% amino acid homology, respectively, suggesting that a variant adenovirus serotype 8 was involved in this outbreak, and is different from the prototype adenovirus 8 virus. This variant had not been detected in Japan prior to 1996 and appears to be the most common adenovirus type 8 involved in cases of epidemic keratoconjunctivitis in Japan at present.