A new cell line derived from a woodchuck hepatitis surface antigen-positive woodchuck hepatocellular carcinoma has been established and named T3-HEP-W1. This new cell line was established directly from a primary woodchuck hepatocellular carcinoma. Adaptation of the cells to the in vitro culture condition was completed after 3 months, with the doubling time of 24 hr. The morphologic features of the cell by light microscopy were of an epithelial type. The modal chromosome number was 100. Ornithine and tyrosine aminotransferase activities were detected. Production of albumin was negative. Integration of woodchuck hepatitis virus DNA was demonstrated by Southern blot analysis, although the secretion of woodchuck hepatitis surface antigen was not detected. T3-HEP-W1 is quite different from the previously reported WH257GE10 cell line and provides another in vitro model for the study of human hepatocellular carcinoma related to hepatitis B virus.