Hepatic nuclear protein has been reported to bind specifically to the TTGGC sequence of the rat regucalcin gene promoter region in stimulating the promoter activity (Misawa and Yamaguchi  Biochem. Biophys. Res. Commun. 279: 275–281). The present study was undertaken to identify transcription factor, which binds to TTGGC motif in the rat regucalcin gene promoter, using the yeast one-hybrid system. The sequence between −525 and −504, which has been defined as a functional promoter element II-b, was used as bait to screen a rat liver cDNA library. Two cDNA clones were identified as a nuclear factor I-A1 (NF1-A1). The results of gel mobility shift assay and mutation analysis using recombinant NF1-A1 protein showed that this protein could specifically bind to TTGGC motif of the II-b oligonucleotide in promoter region. The expression of NF1-A1 mRNA was found in the liver, kidney, heart, spleen, and brain of rats. This study demonstrates that NF1-A1 is a transcription factor in stimulating the rat regucalcin gene promoter activity. J. Cell. Biochem. 84: 795–802, 2002. © 2002 Wiley-Liss, Inc.