CCAAT/enhancer-binding protein (C/EBP) transcription factor family members are related by a high degree of amino acid sequence identity to the basic leucine zipper DNA-binding domain and show distinct but overlapping patterns of tissue- and stage-restricted expression. Although C/EBPalpha and C/EBPbeta have been shown to recognize a consensus sequence derived from regulatory elements in virus and acute-phase response genes, the potential for more subtle differences in the binding preference of the C/EBP family has not been previously addressed. The consensus sequence of C/EBPdelta has not been reported. By using the method of polymerase chain reaction-mediated random site selection to assess the DNA binding specificity of the C/EBP family in an unbiased manner, we demonstrated the sequence preferences for C/EBP family members. With small variations, these C/EBP family members showed similar sequence preferences, and the consensus sequence was identified as RTTGCGYAAY (R = A or G, and Y = C or T). The phosphorylation of C/EBPdelta by casein kinase II increased the binding activity, but did not affect the binding specificity, whereas it was reported that the phosphorylation of C/EBPalpha and C/EBPbeta decreased the binding affinity. The specificity of action of C/EBP family members may be derived from the characteristics of each factor, including the expression profiles, the DNA binding affinities, the cofactors, and so on, in addition to the DNA binding specificities.