In 2005, a serological study was carried out on horses in five ecologically contrasted zones of the Senegal River basin (Senegal) to assess West Nile virus (WNV) transmission and investigate underlying environmental risk factors. In each study zone, horses were randomly selected and blood samples taken. A land-cover map of the five study areas was built using two satellite ETM+ images. Blood samples were screened by ELISA for anti-WNV IgM and IgG and positive samples were confirmed by seroneutralization. Environmental data were analysed using a principal components analysis. The overall IgG seroprevalence rate was 85% (n=367; 95% CI 0·81–0·89). The proximity to sea water, flooded banks and salted mudflats were identified as protective factors. These environmental components are unfavourable to the presence of Culex mosquitoes suggesting that in Senegal, the distribution of the vector species is more limiting for WNV transmission than for the hosts' distribution.