Partially hepatectomized rat hepatocyte nuclear proteins that specifically bind to lactate dehydrogenase A-gene cAMP response element (CRE) were analyzed using Southwestern protein blot analysis. Nuclear proteins with relative molecular masses of 63,000, 58,000, 42,000, 40,000, 37,000 and 14,000 daltons were shown to directly bind to the LDH A-CRE. Compared to the previously identified CRE-binding proteins (CREBs) from PC 12 cells, rat brain, human placenta, and rat hepatocytes, the 14,000 dalton nuclear protein appeared to be a new type of CREB with respect to its molecular size. Southwestern blot analysis, in combination with endogenous protease degradation analysis, revealed that the 14,000 dalton nuclear protein was not derived from the larger CREBs by proteolytic degradation during the preparation of nuclear proteins. Competitive Southwestern blot analysis of hepatocyte nuclear proteins using the LDH A-wild type CRE (wt CRE-1), 8-mer CRE motif (UCRE-1), and a mutant CRE (rat CRE-1) as competitors, suggests that the six hepatocyte CREBs, including 14,000 dalton nuclear protein, bind nucleotide sequences specifically to the LDH-A CRE. The 14,000 dalton nuclear protein is proposed to be a novel type of CREB in regenerating rat liver.