Hepatocyte growth factor activator (HGFA) can activate the single-chain hepatocyte growth factor (HGF) required for embryonic development. We studied the immunohistochemical (IHC) localization of HGFA in adult mouse liver and its developmental changes from embryonic day 12 to postnatal day 30. A heterogeneous distribution of HGFA was observed in adult liver tissues. The hepatocytes around the hepatic veins were preferentially positive for HGFA, whereas those in other areas were negative. Depending on the vascular diameter, the hepatic veins were bordered by a one- to three-cell-thick layer of hepatocytes positive for HGFA, which showed evidence of cell-cell heterogeneity in staining intensity. Immunoelectron microscopy detected ubiquitous distribution of the gold particle reaction product for HGFA in the cytoplasm of these hepatocytes, especially in the rough endoplasmic reticulum. Developmental analysis indicated that there was hardly any staining of HGFA until postnatal day 0 and that noticeable staining was initially detected in the pericentral hepatocytes on postnatal day 3. Subsequently, immunoreactivity increased and the distinct staining pattern had been established by postnatal day 30. These results suggest that HGFA proteins are produced in the hepatocytes surrounding the efferent hepatic veins in the mouse and that development of the unique distributing pattern takes place postnatally.