The antiproliferative effect of heparin on cultured smooth muscle cells in proliferating human smooth muscle cells derived from clinical lesions of intimal hyperplasia was tested. Smooth muscle cells were obtained from stenotic segments excised from failing in situ saphenous vein bypass grafts in three patients. The nonadventitial portion of the excised tissue was explanted into cell culture using standard techniques without the addition of exogenous growth factors. Under these conditions, rapid cell outgrowth was observed from these explants, in contrast to minimal growth of smooth muscle cells from normal veins from the same patients. Immunohistochemical staining with antiactin antibody confirmed that the cells cultured from the stenotic lesions were smooth muscle cells. Incubation of these cells with porcine mucosal heparin revealed a significant (p less than .01) dose-dependent inhibition of cell proliferation as measured by radioactive thymidine incorporation. Mean inhibition of six subcultures tested ranged from 3 to 46%, at heparin concentrations of 1 to 1,000 micrograms/ml. The magnitude of heparin's antiproliferative effect varied among the cell lines from different patients, but 10-30% inhibition was consistently observed at heparin concentrations usually attained in vivo. The maximal inhibition achieved was 65% in one cell line at the highest heparin dose. We conclude that heparin exerts a significant antiproliferative effect on human smooth muscle cells cultured from intimal hyperplastic lesions from in situ saphenous vein bypass grafts.