Feeding male Wistar rats a choline-deficient diet containing 0.07% DL-ethionine (CDE diet) for up to 5 weeks results in the production of two distinct non-parenchymal cell populations, oval and duct-like cells. These cells can undergo replication and display different patterns of expression of glutathione S-transferases (GSTs) and pyruvate kinases (PKs). Oval cells were firstdetected around the periportal region after 1 week of CDE treatment and infiltrated the parenchyma after 2 weeks. Duct-like structures first appeared as isolated ducts in the parenchymal region at 2 weeks and were easily detected after 2.5 weeks. These duct-like structures differed from thebile ducts which reside in the portal region. Large concentrations of duct-like structures in cyst-like clusters were detected after 5 weeks. Enlargement of these structures from single ducts toclusters of up to 20 ducts was observed over 3–5 weeks of CDE treatment. The number of cells forming a duct increased from 5 to 30 cells. We established a double immunocytochemical staining technique to characterize the oval and duct-like cells for their expression of GSTs and PKs. πGST and M₂-PK, which are fetal hepatocytes isoenzymes, are present in virtually all the oval and duct-like cells. Most of the oval cells are devoid of the adult hepatocytes markers, αGST, µGST and L-PK. There are two sub-populations of duct-like cells, one which expresses only fetal markers and the other which co-expresses the adult and fetal isoenzymes. Hence, oval cells display characteristics of fetal hepatocytes and some duct-like cells appear more mature than oval cells. Using a combination of double immunocytochemical and [³H]thymidine labelling techniques we have established that oval cells differentiate into duct-like cells.