We have characterized the nucleic acid binding properties of the major cold shock protein of Bacillus subtilis, CspB. CspB is a member of the cold shock domain (CSD) family, which is widespread among pro- and eukaryotes and shares the nucleic acid binding domain CSD. The CSD domain is highly conserved and binds with strong affinity to the Y-box motif, a cis-element that contains the CTGATTGGC/TC/TAA sequence. In a series of gel retardation experiments using oligonucleotides, which contain the Y-box motif and altered sequences, we show the preferential binding of CspB to single-stranded DNA that contains the ATTGG as well as the complementary CCAAT Y-box core sequence. In contrast CspB exhibits lower affinity to altered Y-box core sequences. Dependent on the length of the oligonucleotide and the degree of sequence deviation from the Y-box core sequence 3- to over 10-fold overexcess of CspB was needed for complete retardation.