In liver tumors induced by chronic WHV infection in the WHV/woodchuck model of HBV infection, activation of genes of the myc family by WHV insertion has been well documented. Several studies have shown that N-myc2 is by far the most frequently involved, and in most cases, its transcriptional activation is due to WHV insertion nearby the gene. N-myc2 has been shown to be also activated by WHV insertion in two downstream loci, b3n and win. Although the extent of insertion in these latter loci in woodchuck tumors has not been investigated, their discovery has led to the notion that therein WHV insertion accounts for N-myc2 activation in the remaining tumors expressing the proto-oncogene in absence of any detectable alteration nearby the gene, a notion remained unproved and not further investigated yet. In the majority of cases, the above observations were derived from tumors developed in colony born laboratory bred woodchucks experimentally infected with standardized viral inocula, mostly of the same lineage. In the present work, we investigated a survey of liver tumors naturally developed in wild woodchucks with naturally acquired chronic WHV infection. Tumors had histological features of well to moderately differentiated HCCs. In most animals, multiple tumor nodules were observed; in the great majority of cases, they were shown to be independent tumors because their WHV integration patterns were not clonally related. 53 independent tumors were investigated for N-myc activation and WHV integration nearby N-myc genes and in the b3n and win loci. Comparison of our results with data from previous studies revealed that, in tumors from naturally infected wild woodchucks, the frequency of WHV integration nearby N-myc2 has a tendency to be lower and, in addition, N-myc2 activation is due to WHV integration nearby the gene significantly less frequently than in tumors from experimentally infected colony born animals (12/28, 43% vs. 15/20, 75%, P = 0.0397). These findings are likely related to the less uniform conditions as to infecting virus and host genetic background in naturally infected wild woodchucks with respect to experimentally infected colony born woodchucks and suggest that viral and/or host factors may influence the site of viral insertion finally detected in overt tumors. In addition, more than one third (11/28, 39%) tumors with activated N-myc2 transcription did not show rearrangement either nearby the gene, or in b3n or in win. These findings challenge the notion that integration in the downstream b3n and win loci is responsible for N-myc2 activation in tumors lacking insertion nearby N-myc2 and suggest that in a considerable fraction of liver tumors, at least from wild woodchucks, N-myc2 activation might be due either to WHV integration in further regions of the N-myc2 chromosomal domain or to other mechanisms related or unrelated to viral insertion.