The efficacy of high-temperature, short-time (HTST) pasteurization (72 degrees C/15 s) when low numbers (< or = 10(3) cfu ml-1) of Mycobacterium paratuberculosis are present in milk was investigated. Raw cows' milk spiked with Myco. paratuberculosis (10(3) cfu ml-1, 10(2) cfu ml-1, 10 cfu ml-1, and 10 cfu 50 ml-1) was subjected to HTST pasteurization using laboratory pasteurizing units. Ten bovine strains of Myco. paratuberculosis were tested in triplicate. Culture in BACTEC Middlebrook 12B radiometric medium detected acid-fast survivors in 14.8% and 10% of HTST-pasteurized milk samples at the 10(3) and 10(2) cfu ml-1 inoculum levels, respectively, whereas conventional culture on Herrold's egg yolk medium containing mycobactin J detected acid-fast survivors in only 3.7% and 6.7% of the same milk samples. IS900-based PCR confirmed that these acid-fast survivors were Myco. paratuberculosis. No viable Myco. paratuberculosis were isolated from HTST-pasteurized milk initially containing either 10 cfu ml-1 or 10 cfu 50 ml-1.