The relative importance of heparin-like compounds in mediating vascular repair is unclear. We investigated how protamine, a chelator of heparin, affected endothelial cell inhibition of vascular smooth muscle cell growth and intimal hyperplasia. The 52% (P < 0.001) reduction in smooth muscle cell proliferation produced by postconfluent endothelial cell-conditioned medium was entirely reversed by pretreatment of medium with heparinase and heparitinase and was inhibited in a dose-dependent fashion by the coadministration of protamine. Pretreatment of conditioned medium with heparinase and heparitinase largely prevented protamine's mitogenic activity, suggesting that protamine affects growth by interacting with heparin-like compounds. Perivascular implantation of polymer-engrafted endothelial cells reduced neointima formation in denuded rat carotid arteries by 92% (P < 0.001) and cell proliferation by 81% (P < 0.001). Coadministration of protamine abolished the inhibitory potential of the cell implants, resulting in a nearly twofold exacerbation of intimal hyperplasia compared with controls (P < 0.001). Thus heparin-like molecules are essential to the biochemical regulation of vascular repair provided by endothelial cells, and the continued routine clinical use of heparin chelators, like protamine, may be questionable.