BACKGROUND: Liver macrophages are a heterogeneous cell population that produces factors involved in fibrogenesis and matrix turnover, including matrix metalloproteinase (MMP) -9. During liver injury, their close proximity to hepatic progenitor cells and the ductular reaction may enable them to regulate liver repair and fibrosis. AIMS: To enumerate and characterise liver macrophages in patients with chronic hepatitis C, to determine whether a distinct population of macrophages is associated with the ductular reaction and portal fibrosis. METHODS: Immunostaining for macrophage markers (CD68, CD163, CCR2), the ductular reaction (keratin-7) and MMP-9 was performed in liver biopsy sections from patients with chronic hepatitis C virus (HCV) (n = 85). RESULTS: Portal tracts were more densely populated with macrophages (10.5 ± 0.36 macrophages/HPF) than lobules (7.2 ± 0.16 macrophages/HPF, P < 0.001) and macrophages were found in close proximity to the ductular reaction. ≥30% of portal and periductal macrophages expressed MMP-9 and these were significantly associated with increasing stage of fibrosis (r(s) = 0.58, 0.68, respectively, both P < 0.001). In contrast, MMP-9(+) macrophages were largely absent in lobular regions and non-diseased liver. Hepatic MMP-9 mRNA levels and gelatinolytic activity were significantly associated with stage of fibrosis (r(s) = 0.47, r(s) = 0.89, respectively, both P < 0.001). Furthermore, a second distinct CCR2(+) macrophage population was localised to the centrilobular regions and was predominantly absent from portal and periductal areas. CONCLUSIONS: These findings demonstrate significant regional differences in macrophage phenotypes, suggesting that there are at least two populations of liver macrophages. We propose that these populations have distinct contributions to the pathogenesis of chronic HCV-related liver disease.