Collagen synthesis is a major function of human intestinal smooth muscle (HISM) cells and contributes to intestinal fibrosis in chronic inflammatory bowel disease. As an extension of previous in vitro studies of the role of heparin in regulating HISM cell proliferation and collagen synthesis, the effect of protamine sulfate was studied. Protamine decreased collagen production by 50% in confluent and proliferating cultures. This effect was concentration-dependent and was selective for collagen in that neither noncollagen production nor DNA accumulation in the culture plates was affected. Other human mesenchymal cells which produce collagen, such as dermal fibroblasts and aortic smooth muscle cells, responded to protamine in a similar fashion. Protamine has a strong cationic charge and is rich in lysine and arginine. To determine which of these properties was important in decreasing collagen production, the effect of protamine was compared to that of other polyionic compounds. Poly-L-lysine decreased collagen production to a lesser degree than protamine. Poly-L-arginine was toxic to the cells. Poly-L-glutamic acid, which has an opposite charge to protamine, had no effect. These findings suggest that both the number and the arrangement of lysyl residues, in addition to positive charge, are important. Binding assays demonstrated that protamine did not inhibit collagen production by binding to ascorbate in the culture medium. Electrophoretic separation and chromatography of collagen types expressed following protamine treatment showed that the ratio of type I to type III collagen remained 2:1. This observation suggests that suppression of collagen production is not specific to a particular collagen type. The selective inhibition of collagen production by protamine provides an important tool to study the regulation of collagen production in human cells and may also provide potential therapy of fibrotic disorders.