Long-term culture of primary hepatocytes derived from normal young male C3H/HeNJcl^MTV+ (C3H) and C57BL/6NJcl (C57) mice, respectively known for very high and low incidences of spontaneous hepatoma, resulted in development of multiple slowly growing epithelial colonies in the C3H case, the number of colonies being increased five-fold when 1.5 mm phenobarbital was added to the culture medium. On the other hand, the primary culture cells from C57 mouse liver gave rise to such epithelial colonies only very rarely, even with phenobarbital.

Immunohistochemical investigation revealed α-fetoprotein and/or albumin production by the colony cells and ultrastructural analysis also revealed some hepatocytic features in them. Subculturing of individual colonies gave rise to cell lines which could be repeatedly passaged. Two of five lines implanted into athymic nude mice manifested tumorigenicity, the resultant neoplasms being diagnosed as a trabecular hepatocellular carcinoma and an adenocarcinoma.

The experimental data suggest that the colony-forming immortal epithelial cells possibly represent early phase precursors of spontaneous mouse hepatocellular carcinomas. This culture system is expected to be useful for future elucidation of the mechanisms underlying spontaneous mouse hepatocarcinogenesis.