Seventeen monoclonal antibodies (MAbs) were prepared against the flavivirus West Nile strain H442. While the majority of these were specific for the major envelope protein, MAbs directed against the NS1 and ns4a nonstructural proteins were also identified. The MAbs were tested by indirect immunofluorescence against 16 southern African West Nile (WN) isolates, representative strains from the two main WN antigenic groups and several viruses from other flavivirus complexes. The MAb reactivities ranged from WN strain-specific to broadly flavivirus-group reactive. Comparison of the local isolates revealed the presence of several different strains, all of which were antigenically distinct from the representative strains of the two WN antigenic groups.