alpha-Fetoprotein (AFP) is a well-established cell differentiation and tumor marker. We showed previously that McA-RH 7777 hepatoma cells are heterogeneous in terms of their AFP cellular expression. In the present study, we developed stable and unstable 7777 hepatoma clones in terms of their AFP phenotype: AFP-producing (AFP+) or AFP-nonproducing (AFP-) clones, and investigated in these clones (a) AFP phenotype related to protein and mRNA levels; (b) cellular morphology; and (c) expression of several liver-specific markers. Our results demonstrated that alpha-albumin expression paralleled that of AFP, from the absence of alpha-albumin message in AFP- clones to high expression in AFP+ clones, suggesting that common mechanisms control the expression of both proteins in this hepatoma cell population. In addition, the karyotypes of the McA-RH 7777 hepatoma cell line and its 15 generated clones were analyzed and correlated to their AFP phenotypes. Only the stable AFP+ clones showed homogeneously staining regions on the chromosome carrying the AFP gene. These results strongly suggest that amplification of either structural or regulatory sequences of the AFP gene is involved in maintaining its high expression.