This study is part of an ongoing analysis of woodchuck hepatitis virus integration sites in the host genome of hepatocellular carcinomas. The study of woodchuck hepatitis virus-DNA integration sites may shed light on the oncogenic mechanisms involved in cellular transformation and tumor formation. Viral integration enhancing cellular proto-oncogene expression is one such mechanism and has been well documented for oncogenic retroviruses such as mouse mammary tumor virus and interleukin-1. By cloning a woodchuck hepatitis virus integration site from a woodchuck hepatocellular carcinoma the authors were able to identify a new member of the myc gene family, N-myc-2. Examination of 30 additional woodchuck hepatomas revealed viral integration commonly occurred near N-myc loci with an additional five woodchuck hepatitis virus integrants near the N-myc-2 gene and one viral integrant near N-myc-1. Three of these N-myc-2 viral integrations were further evaluated and found to be localized within 200 bp of the translation stop codon. This 3′ noncoding region has recently been identified as a common site of murine leukemia virus integration in virally induced T-cell lymphomas and results in increased expression of the N-myc gene. Similar mechanisms can be proposed for hepatocellular carcinoma formation. Woodchuck hepatitis virus integration near cell-growth related protooncogenes, such as N-myc, can juxtapose viral enhancer elements and growth-regulatory genes. Virally induced overexpression of proto-oncogene messenger RNA could result from enhanced transcription or increased messenger RNA stability. To search for such effects the authors analyzed N-myc-2 RNA levels in 30 woodchuck hepatitis virus-related hepatomas. Increased levels of N-myc-2 RNA were found in 18 of 30 tumors, whereas nontumorous portions of the same livers had no detectable N-myc-2 RNA. Taken together these findings suggest that woodchuck hepatitis virus integration can result in altered N-myc-2 gene expression in a significant proportion of woodchuck hepatocellular carcinomas. The deregulation of N-myc gene expression could result in cellular transformation and ultimately tumor formation. Such examples of hepadnavirus-specific oncogenic mechanisms lend credence to theories of hepatitis B virus-induced tumorigenesis and provide models to design molecular investigations of human hepatocellular carcinoma formation.