Cell line WH44KA is a highly malignant woodchuck hepatoma cell line. WH44KA cells contain a single woodchuck hepatitis virus (WHV) DNA integration in the 3' untranslated region of exon 3 of the woodchuck N-myc1 gene. The highly rearranged WHV DNA contains WHV enhancers which activate the N-myc promoter, and a hybrid N-myc1-WHV mRNA is produced, which leads to a high steady-state level of N-myc1 protein. To investigate whether continuous N-myc1 expression is required to maintain the tumor phenotype, we knocked out N-myc expression using a WHV-N-myc1 antisense vector. We identified two WH44KA antisense cell lines, designated 4-5 and 4-11, in which steady-state N-myc1 protein levels were reduced by 95 and 80%, respectively. The growth rates of both antisense cell lines were reduced in comparison to those of wild-type and vector controls. The phenotype of 4-5 and 4-11 cells changed to a flattened appearance, and the cells exhibited contact inhibition. Colony-forming ability in soft agar was reduced by 92% for 4-5 cells and by 88% for 4-11 cells. Cell line 4-11 formed only small, slow-growing tumors in nude mice, consistent with a low level of N-myc1 remaining in the cells. In contrast, 4-5 cells, in which N-myc protein was reduced by greater than 95%, failed to form tumors in nude mice. The integrated WHV DNA contained the complete WHV X gene (WHx) and its promoter; however, we did not detect any WHx protein in the cells by using a sensitive assay. These data demonstrate that N-myc overexpression is required to maintain the malignant phenotype of WH44KA woodchuck hepatoma cells and provide a direct function for integrated WHV DNA in hepatocarcinogenesis.