We developed a culture system from bone marrow-derived neonatal calf macrophages for the assessment of phagocytosis and killing of Mycobacterium paratuberculosis. In the presence of fibroblast-derived macrophage colony-stimulating factor the bone marrow cells were cultured for 8 to 10 days in HMEM medium supplemented with 10% FBS and 5% HS. Conditioned macrophages were infected with M.ptb and subjected to luminol-dependent chemiluminescence. Controls included uninfected macrophages and macrophages infected with Listeria monocytogenes. Oxidative respiratory burst activity was measured in a luminometer and results were recorded via a phagocytosis software program. Maximal intensity of respiratory burst was expressed in mV and obtained in the first 10-12 minutes of the recording cycle irrespective of cycle length and time of cell incubation after infection. Simultaneous treatment with rBoIFN-gamma resulted in a noticeable increase of respiratory burst peak activity during the same time period. Our results suggest that M.ptb. stimulate oxidative bacteriocidal mechanisms during cellular ingestion by bone marrow-derived macrophages which can be augmented by cytokines. A second assay system involved S-methionine labeled M.ptb and labeled Listeria monocytogenes subjected to RboIFN-gamma activated bovine bone marrow-derived macrophages. Short term assays with interferon treated macrophages showed no significant mycobacteriocidal effect by macrophages infected with such labeled M.ptb bacilli. Killing of L. monocytogenes by activated macrophages was noticed. We conclude that infected macrophages primed with interferon need a second signal or longer time intervals for potential mycobacterial killing. The applied isotope incorporation into metabolically active bacilli, however, provides a rapid and sensitive quantitative tool for the assessment of antimycobacterial effects of bovine macrophages.