Forty peptides were synthesized corresponding to hydrophilic clusters of amino acids within the sequences of foot-and-mouth disease virus (FMDV) nonstructural proteins (NSP). Six peptides were studied in more detail and the most promising, a 2B peptide, was evaluated in enzyme-linked immunosorbent assay (ELISA) using sera from naive, vaccinated, and vaccinated-and-challenged cattle as well as bovine sera from field outbreaks. The performance of the new NSP peptide ELISA was compared to that of 4 commercial NSP ELISA kits. Antibody to 2B was detectable from the end of the first week to the second week after infection in most of the nonvaccinated animals and by the second to third week in vaccinated-and-challenged animals. The sensitivity of the 2B peptide ELISA was comparable to the 3ABC Ceditest (Ceditest FMDV-NS, Cedi Diagnostics B.V.; Chung et al., 2002). With some modification and further validation, this 2B test could be useful as a screening or conformational NSP test in postvaccination surveillance for FMD.