The Ras/Raf/MEK/ERK signalling cascade is frequently deregulated in tumourigenic diseases and known to be involved in proliferation and transformation of cells. Also in hepatocellular carcinoma (HCC) increased ERK levels are observed and known to correlate with tumour progression, but the underlying molecular mechanism are unknown. We analyzed expression of Raf-1 kinase inhibitory protein (RKIP) in HCC. Expression of RKIP mRNA and protein was downregulated in HCC cell lines and tissue as compared to primary human hepatocytes (PHH) or non-tumorous liver tissue, respectively. Transfection of an HCC cell line with an RKIP expression construct blocked the Raf kinase pathway resulting in decreased activity of ERK1/2 and AP-1. In contrast, downregulation of RKIP by transfection with an antisense RKIP construct led to increased ERK1/2 and AP-1 activity. Since HCC develop in the majority of cases in cirrhotic liver tissue and cirrhosis is the main risk factor for HCC development, we analyzed RKIP expression also in non-cancerous cirrhotic liver tissues by immunohistochemistry. In contrast to normal liver tissue, where the staining was equally distributed within the cytoplasm, hepatocytes in cirrhotic liver revealed an intense RKIP staining of the membrane. It can be speculated that this changed RKIP expression pattern parallels impaired protein function in PHH in cirrhotic livers that may predispose PHH to malignant transformation. In addition, our study demonstrates functional relevance of downregulation of RKIP in HCC that may play an important role in HCC development and progression.