Owing to frequent overflow of sewage-polluted creeks and rivers in São Paulo city, the population that lives in those surrounding areas is constantly exposed to a high risk of infection by many pathogens. Our aim was to evaluate the occurrence of human adenovirus, especially F species (HAdV-40 and HAdV-41), in sewage and creek water in São Paulo city. A total of 95 water samples were collected from July 1998 to June 1999 and from September 2000 to December 2001 at Sewage Pumping Station Edu Chaves and Creek Pirajussara. Four-litre samples were concentrated by a two-step procedure based on filtration through electropositive filter membrane and ultracentrifugation. Adenoviruses were detected in 69 samples (72.6%) after amplification of a sequence of the hexon gene by PCR. The discrimination of human adenovirus F was done by RFLP. A first screening was performed by restriction of PCR product with TaqI, followed by the discrimination of HAdV-40 and HAdV-41 by RsaI restriction. Sixty-two (89.8%) samples showed F species viruses. Among them, human adenovirus, serotype 41, was the most frequently detected in 17 (62.9%) out of 27 samples and serotype 40 was identified only in 7 (25.9%) samples. No seasonal distribution of adenoviruses was observed.