Mycobacterium paratuberculosis has been cited as the etiologic agent of, or one of the factors associated with, Crohn's disease in humans. Milk has been suggested as a possible source of human infection because M. paratuberculosis is both found in raw milk from infected cows and survives pasteurization. The objective of this study was to measure the decimal reduction time (D-value: time to kill one log concentration of bacteria) for M. paratuberculosis. D-values are an accepted standard measure of thermal tolerance. D-values were measured for multiple strains of M. paratuberculosis suspended in 50 mM lactate buffer (pH 6.8) and milk at 4 temperatures. The D-values were compared with published D-values for other milk-borne bacterial pathogens such as, Listeria monocytogenes, Salmonella typhimurium, and Coxiella burnetii. Viable M. paratuberculosis numbers were quantified using a radiometric culture method (BACTEC). After preheating the menstruum for 30 min., 105-106 cells/ml (final concentration) were mixed with the menstruum in 1.5 ml Wheaton vials, and each vial was sealed and immersed in a water bath adjusted to the target temperature. The D-value of M. paratuberculosis ATCC 19698 (bovine-origin) in lactate buffer at 71°C (D71°C) was 12.9 sec. The D62°C, D68°C, and D71°C values of strain Dominic (human-origin) in lactate buffer were 139, 46, 21, and 13 sec., respectively. These D-values were similar for all M. paratuberculosis strains tested, and higher than those published for Listeria, Salmonella and Coxiella. Coxiella burnetii, the cause of Q fever in humans, is the organism used to set pasteurization time-temperature requirements. These data support the findings of Chiodini et al. that M. paratuberculosis may survive in milk which has undergone pasteurization.