Hepatocellular carcinoma (HCC) is the fifth common type of cancer and third leading cause of cancer deaths in the world. HCC is a genetically heterogeneous disease and multiple pathways and molecules are identified to have role in development of HCC. However, the molecular mechanisms underlying hepatocarcinogenesis are still poorly understood. The transcription factor CCAAT/enhancer binding protein alpha (C/EBPα) is important in differentiation of granulocytes, adipocytes and hepatocytes and mutations in C/EBPα were described in acute myeloid leukemia. C/EBPα is strong inhibitor of cell growth in liver and mouse lacking C/EBPα shows increased proliferation and phenotype resembling HCC. To clarify the role of C/EBPα in hepatocarcinogenesis, we analyzed the genetic alterations of the C/EBPα in HCCs. We have screened C/EBPα mutations in HCC cell lines and tumors by use of sequencing and heteroduplex analysis approaches. In HCC cell lines and tumors, mutations and polymorphisms have been found. Thus, alterations of C/EBPα are involved in hepatocellular carcinoma although it is not frequent. Ours is the first report of C/EBPα alterations in HCC in our knowledge.