Measurement of secreted IFN-γ has proven to be a valuable tool for the detection of animals infected with mycobacterial pathogens, including Mycobacterium avium subsp. paratuberculosis. Previous reports have suggested that tuberculin skin testing can influence the performance of the IFN-γ assay. In the present study, healthy non-infected cows, and cows subclinically and clinically infected with M. paratuberculosis were administered an intradermal injection of johnin purified protein derivative and effects on secreted and intracellular IFN-γ were observed. Significant increases in secreted IFN-γ were noted in antigen-stimulated PBMC isolated from subclinically infected cows within 2 days of the skin test. An increase in intracellular IFN-γ expression was noted after the intradermal injection for all treatment groups after in vitro stimulation of cells with medium only (nonstimulated), or pokeweed mitogen. Intracellular IFN- γ was higher within CD4+ and CD8+ subpopulations for infected cows compared to healthy controls throughout the study. When T cell populations were further defined by CD45RO+ expression, intracellular IFN-γ was higher for CD8+/CD45RO+ lymphocytes compared to CD4+/CD45RO+ cells. These results indicate that intradermal sensitization of cows in the early stage of infection with antigen will enhance the sensitivity of detection by the IFN-γ assay. In addition, CD8+ lymphocytes appear to play an important role as a mediator of M. paratuberculosis infection in naturally exposed cattle.