When alfalfa (Medicago sativa L. cv Apica) plants grown at room temperature are transferred to 2 degrees C, the temperature at which 50% of the plants fail to survive (LT50) decreases from -6 to -14 degrees C during the first 2 weeks but then increases to -9 degrees C during the subsequent 2 weeks. However, when plants are kept for 2 weeks at 2 degrees C and then transferred to -2 degrees C for another two weeks, the LT50 declines to -16 degrees C. These changes in freezing tolerance are paralleled by changes in transcript levels of cas15 (cold acclimation-specific gene encoding a 14.5-kD protein), a cold-induced gene. Cold-activation of cas15 occurs even when protein synthesis is inhibited by more than 90%, suggesting that cold-initiated events up to and including the accumulation of cas15 transcripts depend on preexisting gene products. cas15 shows little homology to any known gene at the nucleotide or amino acid level. The deduced polypeptide (CAS15) of 14.5 kD contains four repeats of a decapeptide motif and possesses a bipartite sequence domain at the carboxy terminus with homology to the reported nuclear-targeting signal sequences. Although the relative amount of cas15 DNA as a fraction of the total genomic DNA is similar in cultivars with different degrees of freezing tolerance, its organization in the genome is different. The possible role of cas15 in the development of cold-induced freezing tolerance is discussed.