Holstein calves were divided into 4 experimental groups: uninfected-unvaccinated controls, unvaccinated-infected, vaccinated-infected, and vaccinated+IL-12 -infected. Vaccinated calves were given commercially available killed M. avium Strain 18 (Mycopar®, Solvay) at 1 week of age by subcutaneous injection. Calves in the vaccine + IL-12 group were given 10 µg human recombinant IL-12 subcutaneously as a separate injection adjacent to the vaccination site. Three weeks after vaccination, calves in infected groups were given an oral challenge of 8 x 10⁹ CFU of field isolate M. paratuberculosis, suspended in milk, once daily for 2 days. Calves were euthanased 3 weeks after oral challenge. Peripheral blood lymphocytes (PBL), prescapular lymph node cells (draining vaccination site), caecal lymph node cells, and spleen cells were cultured in-vitro with M. paratuberculosis antigen. Lymphoproliferation was measured by 3H-thymidine incorporation, and IFN-gamma concentration in culture supernatants was measured by ELISA. Within 3 weeks after vaccination, peripheral blood lymphocytes from vaccinated calves had 3- to 10- fold higher IFN-gamma production compared with unvaccinated calves. Oral challenge without vaccination resulted in no detectable increase in IFN-gamma production by PBLs. Caecal lymph node cells from calves vaccinated+IL-12 produced 4-fold greater concentrations of IFN-gamma than those calves vaccinated without IL-12, 15-fold greater than unvaccinated-infected animals, and 60-fold greater than uninfected-unvaccinated controls. Recombinant IL-12 given with Johne's disease vaccine may enhance the gut associated lymphoid tissue response to M. paratuberculosis.