Two specific carbamyl phosphate synthetase I gene binding nuclear proteins (M. W. 109 kD and 74 kD) have been determined in the rat liver by the protein blotting technique (Southwestern blot assay). The result shows that they are not present in the normal rat spleen and F-26 rat hepatoma cell. The Bal31 nuclease deletion in the CPSI gene 5' upstream region proves that the binding sites for 109 kD and 74 kD are respectively located in the regions of -38 bp to -4 bp and -113 bp to -38 bp. The binding proteins may be the liver-specific ones of the CPSI gene, which are related to hepatocyte differentiation and hepatocarcinogenesis.