Liver regeneration after surgical partial hepatectomy (PH) in retrorsine-exposed rats is accomplished through the outgrowth and expansion of small hepatocyte-like progenitor cells (SHPCs). The cells of origin for SHPCs and their tissue niche have not been identified. Nevertheless, some investigators have suggested that SHPCs may represent an intermediate or transitional cell type between oval cells and mature hepatocytes, rather than a distinct progenitor cell population. We investigated this possibility through the targeted elimination of oval cell proliferation secondary to bile duct destruction in retrorsine-exposed rats treated with 4,4′-diaminodiphenylmethane (DAPM). Fischer 344 rats were treated with 2 doses (30 mg/kg body weight) retrorsine (at 6 and 8 weeks of age) followed by PH 5 weeks later. Twenty-four hours before PH, select animals were given a single dose of DAPM (50 mg/kg). Treatment of rats with DAPM produced severe bile duct damage but did not block liver regeneration. Oval cells were never seen in the livers of DAPM-treated retrorsine-exposed rats after PH. Rather, liver regeneration in these rats was mediated by the proliferation of SHPCs, and the cellular response was indistinguishable from that observed in retrorsine-exposed rats after PH. SHPC clusters emerge 1 to 3 days post-PH, expand through 21 days post-PH, with normalization of the liver occurring by the end of the experimental interval. Conclusion: These results provide direct evidence that SHPC-mediated liver regeneration does not require oval cell activation or proliferation. In addition, these results provide strong evidence that SHPCs are not the progeny of oval cells but represent a distinct population of liver progenitor cells. (HEPATOLOGY 2007.)