Human skin fibroblast culture promises to be a useful system for the investigation of the regulation of collagen biosynthesis and the study of abnormalities in collagen biosynthesis in connective tissue disorders. The effect of culture conditions on procollagen I biosynthesis has been determined. Optimal conditions for collagen biosynthesis were: 10% dialyzed heat-inactivated fetal calf serum, 0.15 mM ascorbate, and 0.078 mM beta-aminopropionitrile. Newly synthesized procollagen I accumulated in the medium at a linear rate for 18 hr. Preincubation of cells in labelling media for 4 hr before adding radioactive proline enhanced synthesis. Collagenase digestion was used to study overall collagen biosynthesis. 94% of all collagen synthesized was found in the medium, and 6% in the cell pellet. Under optimal conditions, collagen comprised 24% of all protein in the medium, and 14% of protein produced by the whole culture.