Although hepatocytes produce collagen in vitro, their contribution to hepatic collagen synthesis in vivo is unknown. To answer this question, we injected rats intraperitoneally with [3H]proline and [14C]ornithine. [3H]Proline labeled prolyl-t-RNA in both hepatocytes and nonparenchymal cells. In contrast, [14C]ornithine was rapidly converted to [14C]arginine via the urea cycle only in hepatocytes, labeling arginyl-t-RNA. Approximately 60% of the 14C in albumin and transferrin was present as arginine while the remainder was found in proline and related amino acids. As expected for proteins that have the same proline/arginine ratio and that are produced solely by the hepatocyte, the [3H]proline/[14C]arginine ratio was very similar in albumin and transferrin. Conversely, in nonparenchymal cells a negligible percentage of 14C was present as arginine. A sizeable percentage of the 14C in hepatic collagen was present as arginine; given the greater proline(+hydroxyproline)/arginine ratio in hepatic collagen, our data indicate that in normal rats, hepatocytes contribute most of newly synthesized hepatic collagen.