Human adenovirus F, serotypes 40 and 41 (HAdV-40 and HAdV-41), are described as a common cause of infantile gastroenteritis. In Brazil, domestic sewage is commonly discharged without treatment into creeks and rivers, especially in the suburban areas. Polluted creeks and rivers, which flow through the city of S¿o Paulo, represent a high risk for public health. Due to frequent overflow of such waters during the rainy season, the population that lives in those surrounding areas is constantly exposed to a high risk of infection by many pathogens. Infectious adenovirus and rotavirus particles have been detected year-round in such waters. Hepatitis A viruses, certainly infectious, were also detected at the same sites. The aim of the present research was to evaluate the occurrence of HAdV-40 and HAdV-41 in raw sewage and creek water in S¿o Paulo city. A total of 43 samples of raw sewage and sewage-polluted creek water were collected from July 1998 to June 1999 at two different sites in the city of S¿o Paulo: Sewage Pumping Station Edu Chaves and Creek Pirajussara, located in the north and south districts of the city, respectively. Four-liter samples were submitted to a two-step concentration procedure based on filtration through a microporous electropositive charged filter membrane (Zeta Plus 60S) and ultracentrifugation. Adenoviruses were detected by PCR using the primers hexAA1885 and hexAA1913, described by Allard et al. (1990), for the amplification of a fragment of 301 bp on the hexon gene. The discrimination of Human adenoviruses F from all other species was done by RFLP. Adenoviruses were detected in 25 samples (58.1%) out of 43 examined. By using TaqI endonuclease, a first screening for specie F viruses was performed and 20 (80.0%) samples showed the presence of serotypes 40 and 41. Fragments of 301 bp could be observed in the same samples, even after prolonged enzymatic digestion, possibly due to the presence of other species of adenovirus than F. Five (20.0%) samples did not show F viruses. The discrimination between HAdV-40 and HAdV-41 was performed by RsaI endonuclease. Human adenovirus, serotype 41, was the most prevalent, being detected in 16 (64.0%) samples out of 25 samples and serotype 40 was identified only in 2 (8.0%) samples. Four samples could not be characterized. No seasonal variation was observed. Includes 20 references, table, figures.