Acclimatization of the psychrotolerant Yersinia enterocolitica after a cold shock from 30°C to 10°C causes transcription of the major cold shock protein (CSP) bicistronic gene cspA1/A2 to increase by up to 300-fold. Northern blot analysis of cspA1/A2 using four probes that hybridize specifically to different regions of CSP mRNA revealed the appearance of a number of cspA1/A2 transcripts that are smaller than the original transcript and transiently visible at the end of the acclimation period. Primer extension and RNA protection experiments demonstrated that these smaller mRNAs have 5′ ends located in the same core sequence (5′-AGUAAA-3′) at five different places within the mRNA, indicating preferential cleavage of the CSP mRNA transcripts. A similar result was obtained for cspB of Escherichia coli, containing two such core sequences. Furthermore, this motif is present in the major CSP genes of a variety of Gram-negative and Gram-positive bacteria. We have therefore termed this sequence cold shock cut box (CSC-box). After inserting a CSC-box into a plasmid-bound lacZ gene in Y. enterocolitica, the mRNA of this construct was cleaved within the CSC-box, and a change in this CSC-box from AGUAAA to AGUCCC dramatically reduced cleavage of the mutated lacZ gene. Mutating all CSC-boxes in Y. enterocolitica of a plasmid bound cspA1/A2 dramatically increases the lag time after a cold shock before re-growth occurs. Based on these results, we suggest that the role of the CSC-box is related to downregulation of cspA mRNA after acclimation to low temperature.