A replication-defective adenovirus 5 encoding foot-and-mouth disease virus (FMDV) capsid and 3C proteinase coding regions (Ad5-FMDV3CWT) was used to vaccinate swine. A single inoculation utilizing 1 x 10(8) plaque forming units (pfu) or an inoculation of 1 x 10(8) followed by a boost of 5 x 10(8) pfu Ad5-FMDV3CWT were tested, along with an inoculation and boost using an adenovirus encoding the FMDV capsid coding region and an inactive form of the 3C proteinase (Ad5-FMDV3CMUT). Sera collected from these animals were examined for the presence of FMDV-specific antibodies using immunoprecipitation, neutralization, and ELISA assays specific for IgM, IgG1 and IgG2. Efficacy studies were performed by placing the vaccinated swine in contact with an FMDV-infected swine and monitoring for signs of disease and changes in serum antibody levels. Ad5-FMDV3CMUT, which is unable to produce FMDV capsid structures, did not elicit FMDV-neutralizing antibodies or protect against FMD. Single inoculation with Ad5-FMDV3CWT generated FMDV-specific neutralizing antibodies, and reduced clinical signs in challenged swine, but failed to completely protect the majority of swine from FMD. Swine which received a primary vaccination with Ad5-FMDV3CWT followed by the boost at 4 weeks generated high levels of FMDV-neutralizing antibodies resulting in complete protection of five of the six swine and limited disease in the remaining animal. Increased efficacy of the two-dose regimen was associated with heightened levels of FMDV-specific IgG1 and IgG2 antibodies.