Foot-and-mouth disease (FMD) vaccines induce antibodies against structural and some nonstructural proteins present in vaccine preparations. To differentiate between FMDV-infected and vaccinated animals, we developed immunochemical assays capable of detecting antibodies against a FMDV nonstructural protein. Recombinant nonstructural 3AB1 protein was expressed in E.coli and in insect cells and used to detect anti-3AB1 antibodies. ELISA and Western blot analysis showed that sera from cattle infected with FMDV reacted with recombinant 3AB1 protein whereas sera from cattle which had been vaccinated against FMDV, mock-infected, or infected with different bovine viruses did not recognize the 3AB1 protein. In contrast, anti-virus infection associated antigen (VIAA) antibodies were present in both FMDV-infected and vaccinated animals. Detection of anti-3AB1 antibodies in sera of experimentally infected cattle obtained between 7 and 560 days postinfection indicated that immunological tests based on the detection of recombinant 3AB1 protein could be used for the diagnosis of FMDV infection.