Tumorigenicity was correlated with levels of expression of the genes for transforming growth factor α (TGF-α), epidermal growth factor receptor, c-myc, c-H-ras,, and c-K-ras in a series of 16 clonally derived transformed liver epithelial cell lines. The clonal lines, which varied in tumorigenicity from 0 to 97%, were established from a phenotypically heterogeneous population produced by repeated exposure of diploid WB-F344 (WB) cells to N-methyl-N′-nitro-N-nitrosoguanidine. Segregation of gene expression with tumorigenicity among clonal lines was determined by correlating rank orders of gene expression by clones relative to expression by wild-type WB cells. Only the expression of the c-myc gene correlated with tumorigenicity among all transformed clones. TGF-α gene expression was not correlated with tumorigenicity among all clones, but it was highly correlated with tumorigenicity among clones that expressed the c-myc gene above the median level for all clones (>5-fold the level of expression by WB cells). Even high levels of expression of the TGF-α gene (up to 60-fold the level of expression by WB cells) were not correlated with tumorigenicity among the clones expressing the c-myc gene at levels <5-fold the level of expression by WB cells. Clones which simultaneously overexpressed both c-myc and TGF-α genes at levels above the median levels for all clones were significantly more tumorigenic than were clones which expressed either or both genes at lower than median levels. These results suggest that overexpressed c-myc and TGF-α genes cooperate in their association with tumorigenicity. Most of the highly tumorigenic clones that overexpressed c-myc and TGF-α also overexpressed the c-H-ras and/or the c-K-ras genes; clones that overexpressed neither of the c-ras genes nor the genes for c-myc and TGF-α were not very tumorigenic, while clones that expressed one or both c-ras genes (but not both c-myc and TGF-α) were variably tumorigenic over an intermediate range.