Collagen formation is an important function of liver parenchymal cells that may be relevant to the pathogenesis of hepatic fibrosis. The types of collagen synthesized by cultured normal rat liver hepatocytes were examined. Cells isolated from adult rats by enzymatic dispersion of the liver were established in primary monolayer culture. Cells were then incubated with radiolabeled proline for 20 hr in the presence of ascorbate and the lathrogen β-aminopropionitrile. Collagen secreted into the cell media was assessed separately from that in the cell layer. The greater proportion of newly synthesized collagen was associated with the cell layer. Collagen types were identified by ion exchange chromatography and by polyacrylamide gel electrophoresis. Types I, III, IV, and V collagen were present in both media and cell layer. Types III and V were the predominant types found. Very little Type I collagen was synthesized by these cultured normal hepatocytes. The percentages of Types I, III, IV, and V collagens, combining media and cell layer, were 6, 38, 19, and 36, respectively.