Freeze-thaw tolerance (cryotolerance) of Listeria monocytogenes is markedly influenced by temperature of growth of the bacteria, and may involve responses to low-temperature stresses encountered during freezing and thawing. A cold-sensitive mariner-based transposon mutant of L. monocytogenes F2365 was found to harbor a single insertion in LMOf2365_1746, encoding a putative RNA helicase, and earlier shown by other investigators to be induced during 4 degrees C growth of L. monocytogenes. The mutant had normal growth at 37 degrees C but completely failed to grow at either 4 or 10 degrees C, and had impaired growth and reduced swarming on soft agar at 25 degrees C. However, the mutation had no discernible influence on the ability of the bacteria to tolerate repeated freezing and thawing after growth at either 25 or 37 degrees C. The findings suggest that the transposon insertion in the putative helicase gene, in spite of the severely cold-sensitive phenotype that accompanies it, does not affect the ability of the bacteria to cope with cold-related stresses encountered during repeated freezing and thawing.