MicroRNAs are small noncoding RNAs that regulate gene expression by targeting messenger RNAs (mRNAs) through translational repression or RNA degradation. Many fundamental biological processes are modulated by microRNAs, and an important role for microRNAs in carcinogenesis is emerging. Because understanding the pathogenesis of viral-associated hepatocellular carcinomas is important in developing effective means of classification, prognosis, and therapy, we examined the microRNA expression profiles in a large set of 52 human primary liver tumors consisting of premalignant dysplastic liver nodules and hepatocellular carcinomas by quantitative real-time polymerase chain reaction. All patients were infected with hepatitis C, and most had liver cirrhosis. Initially, the accessibility of microRNAs from formalin-fixed paraffin-embedded archival liver tissue by real-time polymerase chain reaction assays was shown. Subsequently, target parenchyma from routinely processed tissue was macrodissected, RNA was extracted, and reverse transcription followed by quantitative real-time polymerase chain reaction was performed. Relative quantification was performed by the 2 ؊⌬⌬Ct method with normal livers as a calibrator. In order to obtain a comprehensive microRNA gene expression profile, 80 microRNAs were examined in a subset of tumors, which yielded 10 up-regulated and 19 down-regulated microRNAs compared to normal liver. Subsequently, five microRNAs (miR-122, miR-100, miR-10a, miR-198, and miR-145) were selected on the basis of the initial results and further examined in an extended tumor sample set of 43 hepatocellular carcinomas and 9 dysplastic nodules. miR-122, miR-100, and miR-10a were overexpressed whereas miR-198 and miR-145 were up to 5-fold down-regulated in hepatic tumors compared to normal liver parenchyma. Conclusion: A subset of microRNAs are aberrantly expressed in primary liver tumors, serving both as putative tumor suppressors and as oncogenic regulators.