A continuous cell line was established from a hepatocellular carcinoma obtained from a woodchuck that was sero-positive for woodchuck hepatitis virus (WHV). The cell line, designated WH44KA, grows as an adhering monolayer with a doubling time of 36 hr in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. The cells not only showed epithelial origin on light and electron microscopic examination but also possess biosynthetic markers of the latter, such as albumin and alpha-fetoprotein, which were demonstrated in cultured cells. When they were transplanted into athymic nude mice, tumors developed at the site of inoculation. These tumors were shown to be hepatocellular carcinoma, similar in morphology to the original tumor from which the WH44KA cells were derived. Chromosome analysis revealed a chromosome number ranging from 31 to 126, with a modal number of 35. Integration of WHV DNA was shown by Southern blot analysis. However, WHV surface antigen was not demonstrated in the cultured cells or supernatant medium. The WH44KA cell line appears to be a useful in vitro model for the study of virus-induced hepatocellular carcinoma.