Rat neonatal and neointimal vascular smooth muscle cells differ dramatically from adult medial vascular smooth muscle cells in their growth properties, with the neonatal and neointimal cells exhibiting growth in the absence of exogenously added growth factors. Since it has been hypothesized that extracellular matrix proteoglycans may influence the growth and differentiation of vascular smooth muscle cells, we examined the ability of matrix derived from these cells to influence vascular smooth muscle cell proliferation. To produce test matrices, cells were grown to confluence and removed by brief alkali treatment. Test cells were seeded onto these matrices and the rates of growth in a growth-factor-deficient medium determined. Compared to plastic wells, matrix from neonatal or neointimal cells stimulated the growth of vascular smooth muscle cells. Interestingly, matrix from adult cells was less efficient at promoting growth. Enzymatic digestion of extracellular matrix heparan sulfate, but not of other glycosaminoglycans, further increased the growth-stimulatory effect of extracellular matrix, suggesting that matrix heparan sulfate acts as a growth inhibitor. Consistent with this, biochemical analysis showed that the adult matrix contained a higher percentage of heparan sulfate compared with neonatal or neointimal matrix. These results suggest that autocrine production of heparan sulfate proteoglycans may play an important role in growth regulation of vascular smooth muscle cells during normal vascular development and differentiation as well as in pathological response to injury.