Ninety percent (56/62) of sentinel chickens introduced to two regions within the Italian Alps seroconverted to West Nile virus (WNV) during the summer of 2005, showing a range of antibody titres from 1/20 to 1/320 in a virus neutralization test. Neutralization specificity for WNV antibodies was confirmed on an additional 34 sera that were tested in parallel against WNV (16/34 seropositivity), Usutu virus (3/34 seropositivity) and Koutango virus. The geometric mean neutralizing titre (GMT) calculated for WN-specific antibodies was 33.68 and did not differ significantly amongst sample sites, although the overall results indicate more active circulation of WNV at the higher elevations. Such high levels of seroconversion raise the possibility that many chickens may have been exposed to virus via routes other than mosquito transmission. No chickens or any other local animals were associated with illness due to WNV implying that WNV, and to a much lower extent Usutu virus, circulate harmlessly amongst wildlife species in northern Italy from late May onwards until early autumn.