A spontaneous, pressure-tolerant mutant of Listeria monocytogenes Scott A, designated 2-1, was isolated after several rounds of pressure treatments at 500 MPa for 10 min. Mutant 2-1 was almost 100,000-fold more resistant than the wild type to a pressure of 350 MPa, and about 100-fold more resistant to 450 MPa when pressurized in growth medium. Approximately ten times more mutant cells than wild-type cells survived a 20-min exposure to 55 degrees C, and the mutant appears also to be more resistant to 0.2% H(2)O(2), although the difference could not be confirmed statistically. About 10 times more wild-type than mutant cells survived exposure to growth medium adjusted to pH 2.5 with HCl. The mutant is about 16-fold more sensitive to nisin than the wild type. Mutant 2-1 is non-motile, produces hemolytic activity, is able to grow in fetal calf serum as well as the wild type, and exhibits a lower level of invasiveness of human ileocecal adenocarcinoma cells than the wild type. The mutation in strain 2-1 is a deletion in the ctsR gene that results in the predicted production of truncated CtsR of 20 amino acids compared to a CtsR of 152 amino acids in the wild type. With the exception of its response to pH and possibly also to H(2)O(2), mutant 2-1 shares most of the phenotypes of the previously described ctsR mutant, AK01. The isolation of another spontaneous, pressure-resistant ctsR mutant confirms the central role of this regulatory gene in pressure tolerance of L. monocytogenes. Although such mutants appear of lesser concern to human health then the wild type, current detection methods for Listeria monocytogenes are not able to distinguish between these mutants and wildtype cells.