Cellular analysis of hepatic α-fetoprotein gene expression in normal adult rat and during regeneration induced by partial hepatectomy was performed at the cellular level by in situ hybridization using 35S-labeled complementary DNA probes and immunoperoxidase techniques. In normal adult rat liver sections, a few α-fetoprotein mRNA-cDNA hybrids are detected over all hepatocytes. No protein is detected with routine immunoperoxidase methods. However, after in vivo colchicine blockade of α-fetoprotein secretion, 10 to 20% α-fetoprotein-positive hepatocytes are observed. In regenerating livers, at 2, 6 and 24 hr (before and at the time of the peak of DNA synthesis in the periportal zones), a rise of the nuclear signal level is observed selectively in periportal hepatocytes, without modification of the cytoplasmic signal. At 48 hr (when most hepatocytes have completed at least one replicative cycle), almost all hepatocytes throughout the liver lobule display a rise of the nuclear (2- to 3-fold) and cytoplasmic (1.5- to 2-fold) signal level compared to nonoperated rats. These data show that all hepatocytes in the adult liver express a small number of α-fetoprotein mRNA sequences; they appear to be translated in protein whose secretion can be blocked by colchicine. The moderate increase in α-fetoprotein gene expression induced by liver regeneration takes place in all hepatocytes, in apparently two distinct steps: a very early nuclear accumulation of α-fetoprotein mRNA sequences and a late cytoplasmic accumulation of α-fetoprotein mRNA molecules.