To localize the cellular sources of the collagens excessively deposited in the liver in the course of secondary biliary fibrosis, we have analyzed by in situ hybridization the distribution of alpha 2(I), alpha 1(III), and alpha 1(IV) procollagen and albumin RNA transcripts in rat livers up to 6 wk following common bile duct ligation and scission. In normal liver, moderate amounts of alpha 2(I) and alpha 1(III) procollagen RNA were found in nonparenchymal cells, while alpha 1(IV) procollagen gene expression was at the threshold of detection. Following bile duct obstruction, increasing amounts of alpha 2(I), alpha 1(III), and alpha 1(IV) procollagen gene transcripts were observed in cells of the expanding portal tracts and in perisinusoidal cells in areas of excessive collagen deposition. Procollagen gene expressing perisinusoidal cells were colocalized with desmin-immunoreactive cells, suggesting that Ito cells and transitional cells were among the collagen-expressing cell types. Only alpha 1(IV) procollagen transcripts were found in epithelial cells of newly formed bile ducts. Neither normal nor fibrotic liver showed any hybridization signal above background over hepatocytes, indicating that hepatocytes are unlikely to be a major source of hepatic collagen.