Early detection is vital in controlling the spread of Johne's disease. Cell-mediated immune responses are predominant during the subclinical phase of disease when clinical signs are not evident. The aim of this study was to identify lymphoproliferative responses which may allow the detection of Mycobacterium paratuberculosis (M. ptb.) infection in subclinical stages in sheep. Merino sheep aged 7 months were orally challenged with three doses of 0, 4x10⁴, 4x10⁵ or 4x10⁷ M. ptb./dose (3 animals per group) and samples were taken from peripheral blood and ileal, jejunal and prescapular lymph nodes 15.5 weeks later. Proliferation of peripheral blood mononuclear cells (PBMC) and lymph node cells (LNC) in response to in vitro incubation with medium alone, M. ptb. antigen (10 µg/ml) or Con A (10 µg/ml) for 4 days was determined by flow cytometry. PBMC and LNC were also phenotyped using a panel of antibodies. M. ptb. was not detected by faecal culture in any of the animals and only one microscopic lesion, which had no acid-fast bacilli, was detected in one animal. At this very early stage of disease, proliferation of PBMC increased in response to M. ptb. antigen in two of three animals exposed to 4x10⁵ or 4x10⁷ M. ptb./dose (6 fold and 4.5 fold respectively when compared to the medium only control), but not in the other two groups. Proliferation of LNC in response to M. ptb. antigen was seen in all animals, irrespective of experimental exposure to M. ptb. There was no difference in PBMC phenotype among the different groups. Further studies are being carried out to determine if PBMC proliferation assays can be used to detect Johne's disease at later, pre-clinical stages of disease.