Woodchuck hepatitis virus (WHV) is strongly oncogenic in its native host, producing hepatocellular carcinomas in the majority of chronically infected animals. An important step in this oncogenic process is the activation of N-myc transcription in the liver as a result of integration of viral sequences in cis to the N-myc2 locus. We have examined the viral sequences involved in the up-regulation of N-myc2 using transient transfection assays of permissive HepG2 cells in culture. WHV sequences corresponding to enhancer I of human hepatitis B virus are nearly inactive in N-myc2 activation when tested alone and are not significantly activated by WHV X gene coexpression. By contrast, sequences corresponding to enhancer II strongly activate expression in a position and orientation-independent manner and are only modestly further up-regulated by WHx expression. Qualitatively similar results were observed with sequences of the ground squirrel hepatitis virus (GSHV), which produces hepatomas in woodchucks without insertional activation of N-myc2, but the GSHV EnII element is fourfold less active in N-myc2 up-regulation than its WHV counterpart.