Background. Our aim was to identify differential expression of genes in hepatocellular carcinoma (HCC) with the ultimate goal of discovering novel, diagnostic and therapeutic targets. Methods. We examined differences in gene expression between HCC and noncancerous liver tissue using a cDNA array with probes for 15,843 genes/clones. Two genes, osteopontin (OPN) and S100A6, were found to be >10-fold differentially expressed, and were selected for further immunohistochemical staining in 51 HCC and 10 nonmalignant liver specimens. The relation between OPN and S100A6 alterations and various clinicopathologic parameters was also evaluated. Results. We found a total of 219 genes that were differentially expressed >3-fold. Of these, 109 were upregulated and 110 downregulated. Within this group, 123 genes, including 59 upregulated and 64 downregulated, had been identified previously. These known genes were mainly involved in cell migration, cytoskeleton dynamics, the signaling pathway and cell cycle, and metabolism. OPN expression and S100A6 expression were seen in 26 of 51 (51.0 %) and 16 of 51 (31.4 %) HCC samples, respectively. More importantly, proteins coded by these genes were not found in any noncancerous liver specimen by immunohistochemical analysis. Expression of these genes correlated with poor differentiation (OPN: P = .013; S100A6: P = .008). Conclusion. OPN, a secreted phosphoprotein that has been increasingly implicated in the progression and metastasis of cancer, and S100A6, a member of the S100 protein family that can perform cell proliferation, differentiation, migration, and cytoskeletal dynamics, may be promising diagnostic markers and therapeutic targets for HCC. In addition, the results encourage future studies involving the roles of these proteins in the development and progression of this cancer. (Surgery 2011;149:783-91.)