Hepatocellular carcinoma (HCC) is a common primary cancer associated frequently with hepatitis C virus (HCV). To gain insight into the molecular mechanisms of hepatocarcinogenesis, and to identify potential HCC markers, we performed cDNA microarray analysis on surgical liver samples from 20 HCV-infected patients. RNA from individual tumors was compared with RNA isolated from adjacent nontumor tissue that was cirrhotic in all of the cases. Gene expression changes related to cirrhosis were filtered out using experiments in which pooled RNA from HCV-infected cirrhotic liver without tumors was compared with pooled RNA from normal liver. Expression of approximately 13,600 genes was analyzed using the advanced analysis tools of the Rosetta Resolver System. This analysis revealed a set of 50 potential HCC marker genes, which were up-regulated in the majority of the tumors analyzed, much more widely than common clinical markers such as cell proliferation-related genes. This HCC marker set contained several cancer-related genes, including serine/threonine kinase 15 (STK15), which has been implicated in chromosome segregation abnormalities but which has not been linked previously with liver cancer. In addition, a set of genes encoding secreted or plasma proteins was identified, including plasma glutamate carboxypeptidase (PGCP) and two secreted phospholipases A2 (PLA2G13 and PLA2G7). These genes may provide potential HCC serological markers because of their strong up-regulation in more than half of the tumors analyzed. Thus, high throughput methods coupled with high-order statistical analyses may result in the development of new diagnostic tools for liver malignancies.