Heparin inhibits the proliferation of intimal smooth muscle cells which occurs after denudation of endothelium by air-drying injury in the rat carotid artery. We determined (1) whether the antiproliferative effect of heparin is secondary to effects on platelet adherence to subendothelium or endothelial regeneration and (2) whether the antiproliferative and anticoagulant activities of heparin are related. Morphometric observations by scanning electron microscopy showed that heparin did not alter platelet adherence 5 days after arterial injury and had little or no effect on endothelial regener- ation at 5 and 10 days. To study the relationship between the antiproliferative and anticoagulant effects, we fractionated heparin by affinity chromatography on antithrombin-Sepharose into purified anticoagulant and nonanticoagulant fractions. These heparin fractions were administered to rats in doses which were equivalent either in terms of anticoagulant activity or in terms of mass to the dosage of unfractionated heparin known to inhibit myointimal growth. Additionally, some rats received nonanticoagulant heparin at a dose which was greater in terms of mass than the highest dose of unfractionated heparin which could be administered without inducing fatal hemorrhage. Inhibition of myointimal growth, determined by morphometric analysis of total plaque volume 2 weeks after arterial injury, correlated with total mass of heparin administered but not with anticoagulant activity. Non- anticoagulant heparin given at high dose caused 77% inhibition of myointimal growth(P= 0.02 vs. controls). Heparin inhibition of arterial smooth muscle cell proliferation does not appear to be mediated either by effects on other cells at the level of the arterial wall or by antithrombin. This study should direct attention toward a potential growth regulatory role for arterial glycosaminoglycans.Circ Res 46: 625-634, 1980