Calves experimentally infected with Mycobacterium avium subsp. paratuberculosis and uninfected calves were tested for interferon(IFN)-gamma production after stimulation with purified protein derivative from M. avium subsp. paratuberculosis (PPDp) or a secreted 14 kDa protein (MPP14) specific for the M. avium-intracellulare-scrofulaceum (MAIS) complex. Several calves in both groups responded strongly up to about 5 months to both antigens. Two uninfected calves responded repeatedly, but not always, to MPP14 and PPDp throughout the study. The responses in the uninfected animals seemed to be independent of cell contact between the antigen presenting cells (APC) and the responding population. The supernatant from adherent cells stimulated with MPP14 induced similar levels of IFN-gamma production in CD14+/B-cell depleted peripheral blood mononuclear cells (PBMC) as when the antigen was used directly on PBMC. In contrast, APC/T-cell contact was necessary to induce the IFN-gamma production in infected animals, suggesting that both innate and adaptive IFN-gamma production in response to MPP14 could occur. CD8+ cells contributed to some of the IFN-gamma production in response to MPP14, but the rest could not be explained, while CD4+ cells were responsible for the adaptive response to PPDp. This study showed that secreted proteins could induce innate IFN-gamma production that interferes with diagnostic testing using the IFN-gamma-test.