The cold shock proteins are small peptides that share a conserved domain, called the cold shock domain (CSD), that is important for nucleic acid binding. The Caulobacter crescentus genome has four csp genes that encode proteins containing CSDs. Three of these (cspA, cspB, and cspC) encode peptides of about 7 kDa and are very similar to the cold shock proteins of other bacteria. Analysis by reverse transcription-PCR of the fourth gene (cspD), which was previously annotated as encoding a 7-kDa protein, revealed that the mRNA is larger and probably encodes a putative 21-kDa protein, containing two CSDs. A search in protein sequences databases revealed that this new domain arrangement has thus far only been found among deduced peptides of alpha-proteobacteria. Expression of each Caulobacter csp gene was studied both in response to cold shock and to growth phase, and we have found that only cspA and cspB are induced by cold shock, whereas cspC and cspD are induced at stationary phase, with different induction rates. The transcription start sites were determined for each gene, and a deletion mapping of the cspD promoter region defined a sequence required for maximal levels of expression, indicating that regulation of this gene occurs at the transcriptional level. Deletion of cspA, but not cspD, caused a reduction in viability when cells were incubated at 10 degrees C for prolonged times, suggesting that cspA is important for adaptation to a low temperature.