Eight neutralizing and two non-neutralizing anti-foot-and-mouth disease virus (FMDV) bovine IgG1 and IgG2 monoclonal antibodies (BMAbs) recognize conformationally dependent epitopes. The majority of those shown to neutralize virus passively protected mice from virus challenge, regardless of isotype. Well-characterized anti-FMDV mouse MAbs, representing five independent neutralizing epitopes on O1 serotype virus, were examined with each of the ten BMAbs in a competition-based ELISA. Five of the neutralizing BMAbs (C48, C65, C74, C83 and MH6) were shown to be directed against epitopes on, or in close proximity to, that previously defined as neutralizing antigenic site 2. Another neutralizing BMAb, MH5, bound to an epitope on, or in close proximity to antigenic site 3. Two neutralizing BMAbs (C2 and C96) simultaneously abrogated the binding of mouse antibodies to sites 2 and 4, contesting the autonomous nature of these two regions. None of the BMAbs were shown to be directed towards the immunodominant antigenic site 1. Sequence analyses of neutralization escape mutants supported the competition ELISA results, and included changes at site 2 (VP2 aa C78Y), site 3 (VP1 N46S) and site 4 (VP3 E58K). Additionally, a substitution at a previously unrecorded location (VP2 aa T1881) prevented the binding of site 2 (C48) and sites 2 and 4 (C2) directed BMAbs. Although the bovine and murine anti-FMDV repertoires may not be identical these results support the recognition of similar antigenic features. This is the first report characterizing anti-FMDV MAbs produced from a natural target host, the cow.