The effect of heparin on the biosynthetic phenotype of rat vascular smooth muscle cells (SMC) was investigated in vitro. Addition of heparin to the culture medium of early passage rat SMC resulted in a marked (3-15-fold) increase of a cell layer-associated Mr 60,000 protein that was sensitive to digestion by purified bacterial collagenase and contained significant amounts of hydroxyproline. Pulse-chase analysis of heparin-treated SMC revealed that the Mr 60,000 collagen was a primary and abundant product of these cells and was not processed extracellularly to a smaller form. The inductive effect of heparin could be mimicked by iota carrageenan or dextran sulfates but not by hyaluronic acid, dermatan sulfate, or chondroitin sulfates. The induction was concentration dependent with a maximal effect observed at a heparin concentration of 10 micrograms/ml. Synthesis of the Mr 60,000 collagen increased 18-24 h after addition of heparin to the cultures. Following induction and subsequent removal of heparin, synthesis of the protein remained maximal for at least 12 h and required 72 h to return to a basal level. These data demonstrate that the biosynthetic phenotype of vascular SMC in vitro can be controlled, at least in part, by heparin and related polyanions and suggest a role for similar molecules endogenous to the vessel wall in the regulation of SMC function.