The role of the water cycle in spreading human pathogenic influenza viruses is poorly studied and is not considered to be significant. However, gastrointestinal symptoms developed in a large proportion of influenza A (H1N1) 2009 virus infected people during the pandemic in 2009 and fecal shedding was reported. This fecal route could potentially play a role in the entry of human pathogenic influenza viruses in to the water cycle. Monitoring of influenza viruses in sewage and surface water during the pandemic in 2009 showed that influenza A viruses were detected in sewage and surface water. However, the pandemic influenza A (H1N1) 2009 virus was not detected. These findings imply that the water cycle did not play a relevant role in spreading the pandemic influenza virus during the epidemic in the Netherlands in 2009. Analyses of deliberately contaminated water samples confirmed the ability of quantitative RT-PCR to detect influenza viruses in sewage samples whereas the analysis of large volumes of surface water was strongly hampered by the presence of PCR-inhibiting substances.