Background: Sorafenib is the first agent that has demonstrated an improved overall survival benefit in advanced hepatocellular carcinoma (HCC), setting a new standard for first-line treatment. However, no one has yet been able to predict sensitivity to sorafenib. Pre-treatment pERK level has been shown to be associated with favorable response to such therapy in a phase II clinical study, indicating that pERK may be a potential biomarker for treatment of HCC with sorafenib. Methods: The effects of sorafenib and 5-fluorouracil (5-FU) on cell proliferation were evaluated by cell viability assays in four HCC cell lines (SMMC-7721, MHCC97-L, MHCC97-H and HCCLM6) with different metastatic potential and basal pERK expression levels. Expression levels of pERK were determined by immunocytochemical quantification together with western blot analysis, and pERK density values were also calculated. Correlation analyses were then carried out between the IC50 values of drugs and pERK density values. After basal ERK phosphorylation was down-regulated with U0126 in MHCC97-H cells, cellular responsiveness to sorafenib was assessed by cell viability assay. Results: Basal pERK levels increased stepwise in cell lines in accordance with their metastatic potential. Sorafenib inhibited ERK phosphorylation in a dose-dependent manner in all four cell lines at a concentration between 5 and 20 μM, but the degree of inhibition was significantly different according to their basal pERK expression level (P < 0.0001). In contrast, no significant change was observed after 5-FU treatment. Correlation analyses between the IC50 values and pERK densities revealed that the effects of sorafenib on cell proliferation were significantly correlated with basal pERK levels (Spearman r = -0.8671, P = 0.0003). Resistance to 5-FU was also significantly associated with basal pERK expression in these HCC cell lines (Spearman r = 0.7832, P = 0.0026). After the basal ERK phosphorylation level in MHCC97-H cells was reduced with U0126, they were significantly less sensitive to sorafenib-mediated growth inhibition, with an IC50 of 17.31 ± 1.62 μM versus 10.81 ± 1.24 μM (P = 0.0281). Conclusion: In this in vitro study, pERK was confirmed to be a potential biomarker predictive of sensitivity to sorafenib in treating HCC. The RAF/MEK/ERK pathway may be involved in drug resistance to traditional chemotherapy in HCC. © 2009 Zhang et al; licensee BioMed Central Ltd.