We previously showed that Dlk, a transmembrane protein containing six epidermal growth factor like repeats in its extracellular domain, is strongly expressed in hepatoblasts in murine fetal liver. Here, we examined the expression of Dlk in oval cells, which are adult hepatic progenitors, in the rat 2-acetylaminofluorene/partial hepatectomy (2AAF/PH) model. Reverse transcription polymerase chain reaction analysis showed that Dlk expression was significantly induced in the regenerating liver at day 12 and 14 after PH, when many oval cells were present in periportal areas. Immunofluoresence staining analysis revealed that Dlk(+) cells expressed oval cell markers, cytokeratin 19 (CK19) and alpha-fetoprotein, indicating that Dlk is expressed in oval cells. However, Dlk(+) cells accounted for only about 20% of total CK19(+) oval cells. Dlk(+) cells were localized more distantly from the portal vein than Dlk(-) cells, and were adjacent to mature hepatocytes, though Dlk(+) cells were surrounded by the basal membrane as other oval cells. Furthermore, at day 12 after PH, only 3% of Dlk(+) oval cells expressed Ki67, whereas about 13% of total oval cells expressed Ki67, indicating that Dlk(+) oval cells are less proliferative than Dlk(-) oval cells. Taken together, these results demonstrate that Dlk is expressed in a subpopulation of oval cells and that Dlk(+) cells represent intermediate cells between Dlk(-) oval cells and mature hepatocytes.