The aim of this study was to isolate and characterize potential progenitor cells from healthy dog livers. Stem/progenitor cells can be prospectively isolated from a diversity of tissues using their ability to efficiently pump out the dye Hoechst33342, thereby portraying a side population (SP) in dual-wavelength flow cytometry. We here describe the detection of a SP in dog liver, constituting approximately 3 % of the nonparenchymal-enriched cell fractions. A subpopulation of the SP (approximately 30 %) was immunonegative for the panhematopoietic marker CD45, and consisted predominantly of small, mononuclear, keratin 7-immunoreactive cells; characteristics suggestive of a liver progenitor cell phenotype. Both the CD45- and CD45+ SP showed upregulated expression of progenitor/cholangiocyte marker genes, but also low-level expression of hepatocyte markers, suggesting the presence of progenitor cells committed to the hepatic lineage in both SP fractions. Our findings demonstrate that healthy canine liver contains a small population of cells with progenitor-like characteristics that can be isolated on the basis of efficient Hoechst33342 expulsion.