To estimate the frequency of missed cases of acute West Nile virus (WNV) infection if only WNV RNA or immunoglobulin M (IgM) testing is requested, we measured IgM in specimens negative for RNA and vice versa. Whereas 6 (5.5%) of 110 RNA-negative sera were IgM positive, only 3 (1.0%) of 299 IgM-negative sera were RNA positive (P < 0.05). Similarly, 11 (7.8%) of 141 RNA-negative cerebrospinal fluid specimens (CSF) were IgM positive, but 0 (0%) of 118 IgM-negative CSF were RNA positive (P < 0.05). WNV infections may be missed if only RNA or IgM testing is requested, with a higher frequency of missed cases if only RNA testing is requested.