Cell surface heparan sulfate proteoglycan (HSPG) interactions with type I collagen may be a ubiquitous cell adhesion mechanism. However, the HSPG binding sites on type I collagen are unknown. Previously we mapped heparin binding to the vicinity of the type I collagen N terminus by electron microscopy. The present study has identified type I collagen sequences used for heparin binding and endothelial cell-collagen interactions. Using affinity coelectrophoresis, we found heparin to bind as follows: to type I collagen with high affinity ( Cell surface proteoglycan interactions with type I collagen are likely a ubiquitous mechanism of cell adhesion, yet the interactive sites on these molecules are undefined. Consideration of the complexity of type I collagen structure is relevant to understanding its interactions with heparan sulfate proteoglycans (HSPGs) (see refs. 1 and 2 for review). Type I collagen is secreted as procollagen, which, after proteolytic cleavages, yields the triple-helical monomer composed of two ␣1 and one ␣2 chains. These monomers assemble in a regular staggered fashion into fibrils, which display the repeating D-period pattern, consisting of fine crossfibril bands (called the a, b, c, d, and e bands) in positively stained electron microscopy preparations.Triple-helical type I collagen conformation is necessary for its high-affinity binding to HSPGs, or to heparin, a chemical analog of its heparan sulfate chains (3-5). Furthermore, the C-terminal triple-helical fragment of type I collagen, generated by vertebrate collagenase treatment, showed a higher affinity for heparin than the did the N-terminal fragment (4).To localize more precisely the heparin-binding regions on type I collagen, we studied complexes between collagen monomers and heparin-albumin-gold particles by electron microscopy (6), and we observed heparin binding primarily to a region on the triple helix near the procollagen N terminus. In collagen fibrils, heparin-gold bound to the a bands region within each D-period, which is consistent with an N-terminal heparinbinding site on its monomers. The resolution of the mapping technique was insufficient to assign heparin-binding function to any particular protein sequence. Thus, amino acid sequences were inspected in relation to the heparin-binding locations observed by electron microscopy, searching for basic domains that might be suitable as heparin-binding sites. A highly basic sequence was found near the procollagen N terminus, and within the a bands fibril region, corresponding to amino acid residues 87-92 of the rat ␣1 chain. To test the heparin-binding function of this or other sites of type I collagen, here we have studied how mimetic triple-helical peptides (THPs) including various collagen sequences interact with heparin. We have also explored the function of these sequences in endothelial cell interactions with type I collagen during endothelial tube formation.