In aromatic amine-induced rat hepatomas, the aldehyde dehydrogenase (AIDH) phenotype is qualitatively and quantitatively different from that of normal liver. To identify the mechanism(s) underlying the expression of the tumor-specific AIDHs, we have followed the time course of appearance of the new phenotype during hepatoma formation in Sprague-Dawley rats following brief dietary exposures to 2-acetylaminofluorene (0.02%; 32 days). Tumor promotion by phenobarbital (0.05% in the diet) was also used to compare the effects of a variety of tumor induction protocols on the AIDH phenotype. No change in the AIDH phenotype is detectable by total activity assay, gel electrophoresis, isoelectric focusing, or immunochemical methods during or following exposure to carcinogen or promoter until tumors are grossly observed in liver. Concomitant with tumor appearance, the tumor-specific AIDH phenotype appears. The phenotypic change is limited to the tumor; morphologically and histologically normal liver directly adjacent to the tumor and normal lobes of a tumor-bearing liver do not possess the tumor AIDH phenotype. No correlation exists between tumor size and the degree of deviation of the AIDH phenotype from normal. Nor is there any correlation between the degree of AIDH phenotype deviation and the histology of the various tumors observed. We conclude that the tumor-specific AIDH phenotype is not associated with altered liver metabolism due directly to carcinogen or promoter exposure. Rather, the mechanism of this phenotypic change requires that transformation-associated, stable genetic changes occur in the cells affected by carcinogen that are later expressed as the altered AIDH phenotype.