Several structural analogues of proline have been shown to be incorporated into proteins in place of proline. As a consequence, the proliferation of cells in culture and the extracellular deposition of collagen in animal systems are reduced. In this study, the effects of two proline analogues, cis-4-hydroxy-L-proline and L-azetidine-2-carboxylic acid, on the growth parameters and procollagen production by cultured normal human skin fibroblasts were examined. The results indicated that incubation of the cells with the analogues reduced the rate of fibroblast proliferation and lowered the plating efficiency. Further experiments demonstrated that fibroblasts in the presence of L-azetidine-2-carboxylic acid synthesized procollagen polypeptides which were not in a triple-helical conformation, as judged by limited pepsin proteolysis. Also, a significantly increased fraction of the newly synthesized collagenous peptides was in a dialyzable form, suggesting increased degradation of the nonhelical chains. The rate of translation of collagenous polypeptides and the preprocollagen messenger RNA activity in the cells were not affected by the analogues. The proline analogues thus appear to inhibit the production of procollagen on the posttranslational level by preventing the polypeptides from folding into a stable triple-helical conformation. The nonhelical polypeptides are then readily susceptible to proteolysis leading to reduced deposition of extracellular collagen fibers. Similar experiments were also performed with fibroblasts cultured from patients with active progressive systemic sclerosis. Quantitatively and qualitatively comparable inhibition of procollagen production by L-azetidine-2-carboxylic acid was noted with scleroderma cells as with control fibroblast cultures. The results suggest, therefore, that proline analogues may, in the future, prove useful in limiting excessive collagen deposition in scleroderma and other forms of dermal fibrosis.