The cellular sources of collagen in normal rat liver have been examined. Hepatocytes and nonparenchymal (sinusoidal) cells were isolated and established in primary monolayer culture. These cells were incubated with radiolabeled proline in the presence of l-ascorbate and beta-aminopropionitrile. Nondialyzable material was prepared from the cell layer and the medium from each type of culture. The level of collagen accumulation was determined by measuring labeled hydroxyproline and sensitivity to purified bacterial collagenase. In hepatocytes, collagen represented 0.2% of both secreted and cell-associated labeled protein. In sinusoidal cells, collagen was 3.2% of secreted and 1.1% of cell-associated proteins. The total secreted labeled collagen, expressed per microgram of DNA, was similar in hepatocytes and sinusoidal cells. However cell-associated collagen in hepatocyte culture was approximately 10-fold that present in sinusoidal cells. These findings indicate that, while collagen formation is a relatively important function of sinusoidal cells, in normal liver the contribution of hepatocytes to total hepatic collagen accumulation may be substantial.