Nuclear factor 1 (NF1) family proteins, which are encoded by four different genes (NF1-A, NF1-B, NF1-C and NF1-X), bind to the palindromic sequence and regulate the expression of many viral and cellular genes. We have previously purified NF1-A and NF1-B from rat liver as factors that bind to the silencer in the glutathione transferase P gene, and have also reported the repression domain of NF1-A. In the present study we cloned five cDNA species (NF1-B1, NF1-B2, NF1-B3, NF1-C2 and NF1-X1) and compared their expression profiles and the affinity and specificity of the DNA binding of these NF1 family members. By Northern blot analysis, we found that the expression profiles of the NF1s are indistinguishable in the various tissues of the rat. The DNA-binding affinities of NF1-A and NF1-X are higher than those of NF1-B and NF1-C, whereas all four NF1 proteins showed the same DNA-binding specificity. Transfection analyses revealed that the function of NF1-B on the transcriptional regulation differed between NF1-B isoforms and was affected by the factor(s) that bind to the promoter regions. In addition, we identified the transcriptional regulatory domain of NF1-B, which is enriched with proline and serine residues.