West Nile Virus (WNV) may cause significant morbidity and mortality in birds, humans and horses. WNV is not (yet) present in the Netherlands, but it is steadily approaching from south-eastern Europe. Recently, a WNV-vaccine (Duvaxyn ® -WNV) became available in Europe. It is claimed that vaccination results not or only infrequently in an IgM response making it possible to differentiate acutely-infected horses from vaccinated horses by using an IgM-based ELISA. The aims of the study were to investigate the supposition that vaccination does not result in IgM production, to evaluate whether different intramuscular injection sites influence the immunological responses and whether any local or systemic adverse reactions would occur. Twenty horses and ponies, 3 to 21 years old, were divided into four groups and horses of each group were vaccinated twice (day 0 and 21) at different intramuscular sites (neck, pectoral muscles, rump and thigh). Weekly blood samples were collected over a period of 42 days and tested for Flavivirus IgM antibodies using a WNV-IgM ELISA and for Flavivirus Ig antibodies using a WNV blocking ELISA. None of the horses tested positive for WNV antibodies prior to vaccination. All horses showed a clear Ig (total antibody) response to the WNV vaccination, but in two horses this response was limited. Surprisingly, ten horses also gave a (limited) positive IgM response. This suggests that an IgM capture ELISA will not distinguish horses with an acute WNV infections from recently vaccinated horses with certainty. The location of the intramuscular injections had no significant effect on the immunogenic response. No systemic reactions were encountered nor were there local reactions at any of the injection sites.