The objective of this study was to compare the production of gamma-interferon (IFN-g) in response to Avian and Johnin Purified Protein Derivatives (PPD) in blood and biopsies of prescapular lymph nodes (PLN) from sheep infected experimentally with Mycobacterium paratuberculosis. Six of 12 lambs, aged from 1.5 to 2 months, were inoculated orally with M. paratuberculosis. The other six non-inoculated lambs constituted negative controls. Twelve months after inoculation blood for an agar gel immunodiffusion test (AGID), enzyme-linked immunosorbent assay (ELISA) and for the IFN-g assay were collected, and sheep were humanely killed. After euthanasia, punch biopsies of the PLN for the IFN-g assay and intestinal and mesenteric lymph node samples for histopathological examination and culture were obtained from each sheep. Samples of blood and PLN biopsies were stimulated with two antigens; Avian and Johnin PPD. Among the six sheep dosed with M. paratuberculosis, four had bacteriological evidence of infection, of which two had unequivocal paratuberculosis lesions. The PLN and blood IFN-g assays gave positive reactions in six inoculated animals, regardless of the type of antigen used as a stimulus. In comparison, the AGID and ELISA detected three and four inoculated sheep, respectively. None of the six non-inoculated sheep had histological evidence of paratuberculosis or tested positive by culture, AGID, ELISA or the IFN-g assay on samples of blood stimulated with Johnin PPD. One non-inoculated animal tested positive in the Johnin PPD PLN IFN-g assay. The blood IFN-g assay and PLN IFN-g assay in which Avian PPD was used as an antigen gave positive reactions in two and three non-inoculated sheep, respectively. Differences between IFN-g responses in blood and PLN biopsies to Avian PPD and Johnin PPD are discussed.