Low temperature is an important environmental factor influencing plant growth and development. In this study, we report the characterization of a genetic locus, HOS2, which is defined by three Arabidopsis thaliana mutants. The hos2-1, hos2-2 and hos2-3 mutations result in enhanced expression of RD29A and other stress genes under low temperature treatment. Gene expression in response to osmotic stress or ABA is not affected in the hos2 mutants. Genetic analysis indicates that the hos2 mutations are recessive and in a nuclear gene. Compared with the wild-type plants, the hos2-1 mutant plants are less capable of developing freezing tolerance when treated with low non-freezing temperatures. However, the hos2-1 mutation does not impair the vernalization response. These results indicate that HOS2 is a negative regulator of low temperature signal transduction important for plant cold acclimation.