Absence of Kupffer cells in rat liver hyperplastic nodules induced by a chemical carcinogen was demonstrated by intravenous injection of indian ink. Hyperplastic nodules appeared 4 weeks after diethylnitrosamine (DEN) was administered, and the nodules continued growing and became eosinophilic hyperplastic nodules after 5 to 6 weeks. After intravenous injection of indian ink, hyperplastic nodules were observed as carbon-free white nodules, which were macroscopically distinguishable from the black surrounding tissue. As observed by light microscopy, Kupffer cells were absent in hyperplastic nodules in contrast to being present in the surrounding tissue. Scanning electron microscopy confirmed these findings and furthermore revealed that the sinusoidal endothelium of hyperplastic nodules had no fenestrae. Injection of indian ink is a useful method for delineation and enucleation of hyperplastic nodules in the study of morphological and chemical changes of nodules.