To quantify cellular immunity in ruminant paratuberculosis, parameters like gamma-IFN release and lymphocyte proliferation have been measured after in vitro stimulation with specific antigen. In this study we describe the quantification of surface interleukin 2 receptor (IL-2R), an early marker of lymphocyte activation and a prerequisite for lymphocyte proliferation, after in vitro stimulation with PPD Johnin. In order to characterise the phenotype of the lymphocytes expressing IL-2R, cells were processed for dual- or three colour analysis by flow cytometry. Blood collected from Mycobacterium avium ss.paratuberculosis inoculated and healthy goats were diluted 1:10 in RPMI and stimulated with 10 mg/ml PPD Johnin for 72 hours. Non-stimulated control cultures were included. Erythrocytes were hypotonically lysed and leukocytes stained with monoclonal antibodies against IL-2R and CD4 or IL-2R, CD8 and gamma-delta-T cells using FITC, PE and Chycrome conjugated secondary antibodies followed by flowcytometric analysis. Small lymphocytes and lymphoblasts were distinguished by their forward/side scatter characteristics. In both regions the population of IL-2R expressing cells were determined as CD4+, CD8+, gd+ or CD8+/gd+ . At ten months post oral infection, the percentage of IL-2R expression of small lymphocytes and lymphoblasts were significantly higher in the infected group of animals than in the control group. In the lymphoblast region, there were significantly more IL-2R positive cells expressing CD4+ and CD8+ among the infected animals, while the main population of IL-2R expressing cells in the control group were of the gamma-delta-T cell subset. In the region of small lymphocytes there were significantly more IL-2R cells expressing CD8+ among the infected animals.