To analyze the cellular mechanisms of alpha-fetoprotein (AFP) gene activation during liver regeneration, we have measured the steady-state level of liver AFP mRNA by dot blot and revealed AFP transcripts and protein in liver sections by in situ hybridization and immunoperoxidase. AFP gene activation was studied from rats intoxicated with carbon tetrachloride or D-galactosamine, two toxics inducing different patterns of liver necrosis and regeneration. At 48 and 72 hours of carbon tetrachloride intoxication, liver AFP mRNA steady-state levels were slightly increased. On liver sections, large necrotic centrilobular areas were apparent with a 7- to 10-fold increase of hepatocyte mitotic index as compared with controls. At the same times of intoxication, a 2-fold increase in the signal level for AFP mRNA over all nonnecrotic hepatocytes was observed, whereas the protein remained undetectable in most of the hepatocytes. In contrast, after D-galactosamine intoxication, no increase of AFP mRNA steady-state levels was detectable. Scattered necrotic hepatocytes were visible at 48 and 72 hours of intoxication, with only a 2-fold increase of hepatocyte mitotic index, as compared with controls. Moreover, while the hybridization signal level for AFP mRNA was not increased over hepatocytes, large amounts of AFP mRNA and protein were detected in proliferated oval cells and bile duct-like structures. These results suggest that at least two distinct mechanisms at the cellular level may explain AFP gene activation during chemically-induced liver regeneration: (a) a moderate increase of AFP mRNA by all the remaining hepatocytes probably linked to their reentry into the proliferative cycle, which appears the main mechanism after carbon tetrachloride intoxication, and (b) a quantitatively important AFP expression by a small number of proliferated cells with new phenotypes (oval cells and bile-duct like structures), observed in the D-galactosamine model.