A study was undertaken to determine the efficacy of HTST pasteurization (71.7°C/15 s) when low levels of Mycobacterium paratuberculosis are present in milk. The levels of M. paratuberculosis employed in this study are considered more likely to be encountered in milk from cattle with Johne's disease than those employed in our previous study (10⁸ and 10⁴ CFU/ml). Samples (250 ml) of aseptically-drawn cows' milk were inoculated with low numbers (10³ CFU/ml, 10² CFU/ml, 10 CFU/ml or 10 CFU/50 ml) of M. paratuberculosis and subjected to HTST pasteurization using Franklin plate heat exchangers. A total of nine bovine strains of M. paratuberculosis were tested in triplicate at each of the inoculum levels. Where necessary, low numbers of the organism were concentrated prior to enumeration by centrifuging 10 or 50 ml aliquots of milk (2400 x g, 20 min) and resuspending the pellet of cells in 1 ml of PBS. The most probable number of M. paratuberculosis present in the milk before and after pasteurization was estimated by inoculating slopes of Herrold's egg yolk medium containing mycobactin J (HEYM). BACTEC Middlebrook medium supplemented with sterile egg yolk and mycobactin J was also inoculated. Both media were incubated at 37°C for up to 18 weeks and any growth observed was tested for acid-fastness by the Ziehl-Neelsen strain. To date, suspect acid-fast survivors have been observed in a number of the HEYM and BACTEC cultures of HTST pasteurized milk. The acid-fast bacteria were generally present along with other non-acid-fast bacteria arising from the milk. Attempts are being made to decontaminate these cultures prior to IS900-based PCR testing which will confirm whether these suspect survivors are M. paratuberculosis.