The adjuvant effect of porcine interferon alpha (pIFN-alpha) was examined in swine vaccinated with a replication-defective adenovirus containing foot-and-mouth disease virus (FMDV) A24 capsid and 3C proteinase coding regions (Ad5-A24). Groups of swine were inoculated with either high or low doses of Ad5-A24 in the presence or absence of Ad5-pIFNalpha or with a control Ad5 and challenged by intradermal inoculation in the heel bulb with FMDV at 42 days post-vaccination. After challenge all control animals developed viremia and lesions. Animals receiving low-dose Ad5-A24 had similar clinical disease, but only three of five animals developed viremia, while addition of IFN resulted in a delayed onset of lesions in three animals and only one animal had detectable viremia. Animals vaccinated with high-dose Ad5-A24 had no viremia, significantly fewer lesions and delayed onset of disease compared to the control and low-dose vaccine groups. Four of five pigs vaccinated with high-dose Ad5-A24 plus IFN were completely protected from disease and only one animal had a lesion which was restricted to the site of challenge. Thus, pIFN-alpha enhances the long-term level of protection induced by the Ad5-FMD vaccine, supporting its use as a potential adjuvant in FMD vaccination strategies.