Prolonged culture of hepatocytes isolated from mouse liver results in the spontaneous development of colonies of liver epithelial cells that can proliferate indefinitely in vitro. We established 5 such cell lines from C3H/HeJ mice (C3H) and 22 cell lines from C3H/HeJ × C57BL/6J F1 mice (C3B6F1) to investigate whether any specific karyotype alterations may be associated with the development of such cells. These lines retained some properties of hepatocytes as well as showing bile-duct-cell characteristics, and comprised mainly near-diploid and/or hypotetraploid cells. Karyotypic analysis of the C3H cell lines indicated that most cells have loss of chromosome 4 or deletion involving the C7 portion, while at least I (for near-diploid cells) or 2 (for hypotetraploid cells) copies of chromosome 4 were usually intact. In addition, gain of an extra chromosome 15 was frequently observed in these cell lines. Analysis of the microsatellite DNA polymorphic markers in 22 C3B6F1 lines revealed that a majority of them showed loss of heterozygosity (LOH) for, at least, 1 of 3 polymorphic loci on chromosome 4, but not for 2 loci on chromosomes 7 and 11. Mouse chromosomes 4 and 15, therefore, may contain genes related to the ability of such liver epithelial cells to grow indefinitely in vitro [The locus on chromosome 4 was designated as liver-cell immortalization (LCI) locus].