Neovascularization is important in the development of liver metastasis. We sought to define the origin and fine structure of the blood supply of small experimental liver metastases in rats using an injection replica method. Liver metastases were produced by intraportal inoculation of ascitic fluid containing AH60C heparoma cells in male Donryu rats (n = 40). Intrahepatic microvasculature was studied by scanning electron microscopy and by stereomicroscopy of microvascular casts produced by perfusion via the abdominal aorta or portal vein 7 days following tumor inoculation. Intrahepatic microvasculature in rats without liver metastases (n = 10) also was studied by scanning electron microscopy. In the normal liver, branches of the hepatic artery typically terminated in the peribiliary plexus and less frequently led to sinusoids and terminal portal veins. In 69 metastatic tumors ranging from 269 to 1875 μm in diameter, arterially perfused metastatic tumors larger than 300 μm showed newly developed neovascularization. Portally perfusion of metastatic tumors did not visualize neovascularization irrespective of tumor size. At the periphery of metasrases, tumor vessels disclosed by arterial perfusion most often communicated with the peribiliary plexus and less frequently with terminal arterioles. Metastatic liver tumors as small as 300 μm in diameter receive their main blood supply from the hepatic artery but not from the portal vein, and tumor vessels more often are derived from the arterially filled peribiliary plexus rather than from terminal arterioles. (C) 2000 Academic Press.