Effectiveness of heat inactivation during processing and the concentration of Mycobacterium paratuberculosis in raw milk have been identified in quantitative risk analysis as the most critical factors contributing to the possible presence of viable M. paratuberculosis in dairy products. Laboratory simulations of pasteurisation have suggested that increasing the withholding time would enhance the likelihood of killing M. paratuberculosis during processing. A quantitative assessment of the lethality of pasteurisation was undertaken using a sensitive culture technique with minimal decontamination treatment capable of detecting one organism per 10 ml. M. paratuberculosis was artificially added to raw whole milk which was then homogenised and pasteurised in a pilot industrial pasteuriser designed for research purposes to simulate a commercial continuous flow milk pasteuriser. The holding tubes were tested to ensure pasteurisation times of 15 s, 20 s and 25 s with a flow-rate of about 3000 l/hr and a Reynold's number of 62,112. The heat exchanger was tested prior to use to confirm there was no leakage and the inlet and outlet hold-tube temperatures and flow-rate were monitored every 10 s during operation of the plant. Twenty batches of milk containing 10⁶ to 10⁷ organisms/l were processed with various temperature and time combinations of 72-78°C and 15-25 s. Thirty 50 ml milk samples from each processed batch were cultured and the logarithmic reduction in M. paratuberculosis was determined. In 17 of the 20 batches no viable M. paratuberculosis were detected representing greater than 6 log10 reductions. These experiments were conducted with very heavily artificially contaminated milk to facilitate the measurement of the logarithmic reduction. In 3 of the 20 batches of milk pasteurised at 72°C for 15 s, 75°C for 25 s and 78°C for 15 s, a few organisms were detected. Pasteurisation in these experiments was found to be very effective in killing M. paratuberculosis with a logarithmic reduction in all batches greater than 4 log10.