The proliferation of vascular smooth muscle cells (VSMC) plays a significant part in both the developing atherosclerotic lesion and in restenosis. Heparin has been widely reported to inhibit the growth of VSMC in culture and intimal VSMC in some animal models of vascular hyperplasia. Clinical trials with heparin, however, have failed to inhibit restenosis following angioplasty. Bovine serum is normally used as a growth supplement in in vitro VSMC growth assays. We have compared the effects of human serum with those of bovine serum on the cellular response to heparin in human VSMC culture. While heparin inhibited the proliferation of human VSMC in the presence of bovine serum, it was totally ineffective in the presence of human serum. These observations were consistent over a wide range of serum and VSMC samples. Experiments utilizing neutralizing antibodies to a number of growth factors showed that cells in either serum were similarly dependent on platelet-derived growth factor for proliferation. In contrast, proliferation in the presence of bovine serum was shown to be dependent on extracellular basic fibroblast growth factor, whereas that in human serum was not. Direct binding of [<sup>3</sup>H]-heparin to VSMC was significantly reduced in the presence of human serum compared with bovine serum, and the former contained twice the concentration of heparin-binding factors of the latter. Removal of heparin-binding factors from either serum type significantly reduced the proliferation potential. Fractionation of heparin-binding factors from human serum showed that the major growth-promoting activity, together with heparin resistance, was contained within a fraction excluded by a 100,000 molecular weight membrane. We conclude that the mechanism of resistance to heparin in human serum is likely to be due to a combination of differential growth factor binding and interference with heparin interaction with cellular receptors by a high molecular weight heparin-binding factor. This phenomenon may significantly contribute to the lack of success of heparin as an antirestenotic agent in clinical trials.