Stem cell‑derived hepatocyte precursor cells represent a promising model for clinical transplantation to diseased livers, as well as for establishment of in vitro systems for drug metabolism and toxicology studies. The present study aimed to establish a new method of induction of hepatocyte differentiation using various factors and evaluate the effect of different partial hepatectomies and the duration of collagenase perfusion on hepatic stem cell proliferation and differentiation. A rat model of hepatic oval cell proliferation was established by partial hepatectomy (PH). Following 73.1 and 83.4% PH, rats underwent perfusion with IV collagenase for 10, 20 and 30 min. Density gradient centrifugation was performed and cells in the supernatant were cultured in various combinations of factors to induce oval cells to differentiate into mature hepatocytes. Cells were characterized for hepatocyte marker expression by morphology, flow cytometry, immunoﬂuorescence and western blot analysis. Hepatic oval cells isolated from rats at 7 and 14 days post‑PH exhibited properties of hepatic stem/progenitor cells. Following culturing in RPMI‑1640 medium with hepatocyte growth factor and fibroblast growth factor‑4, the cells resembled primary human hepatocytes with regard to morphology and expression of the hepatocyte markers, cytokeratin 18 (CK‑18) and α‑1‑fetoprotein (AFP). Optimal differentiation of hepatic stem cells to CK‑18‑ and AFP‑positive cells was observed when stem cells isolated from 83.4% PH rats (7 days following surgery) were perfused with IV collagenase for 20 min. The results of this study provide novel insights into characteristics of rat hepatic stem cells.