Adult liver functions are regulated by several hepatocyte nuclear factors (HNFs). HNF4alpha and HNF1alpha are involved in metabolic functions in the liver. The expression of proline oxidase (PO) and proline dehydrogenase was downregulated in the HNF4alpha liver-specific null mice. In addition, the expression of PO was also diminished in the liver derived from HNF1alpha-null mice. The -160 bp proximal promoter region of the PO gene has two HNF4alpha- and HNF1alpha-binding consensus sites. Transactivation, electrophoretic mobility shift and chromatin immunoprecipitation studies revealed that these regions are important for PO promoter activity. These results suggested that HNF4alpha and HNF1alpha regulate proline metabolism in adult liver.