Fibroblast Growth Factor-2 (FGF2) is a major inducer of neovascularization (angiogenesis). Heparin activates FGF2 by favoring formation of ternary complexes with its cellular receptors (FGFRs). Controlled 2-O-desulfation followed by exhaustive periodate oxidation/borohydride reduction has been used to generate sulfation gaps within the prevalent heparin sequences, building-up arrays of pentasulfated trisaccharides (PST, consisting of a 2-O-sulfated iduronic acid flanked by two N,6-disulfated glucosamines) spaced by reduced, glycol-split uronic acid (sU) residues. The structure of the prevalent sequences of the novel heparin derivative has been confirmed by mono- and two-dimensional NMR analysis. NMR spin-lattice relaxation times (T2) and nuclear Overhauser effects suggest that the sU residues act as flexible joints between the PST sequences and cause a marked distortion of the chain conformation of heparin required for formation of ternary complexes. Since the splitting reaction also occurs at the level of the essential glucuronic acid residue of the active site for antithrombin, the heparin derivative has no anticoagulant activity. However, it fully retains the FGF2-binding ability of the original heparin, as shown by its capacity to protect FGF2 from trypsin cleavage and to prevent the formation of heparan sulfate proteoglycan (HSPG)/FGF2/FGFR1 ternary complexes. However, when compared to heparin it showed a reduced capacity to induce FGF2 dimerization and to favor the interaction of [125I]FGF2 with FGFR1 in HSPG-deficient, FGFR1-transfected CHO cells. Accordingly, it was more effective than heparin in inhibiting the mitogenic activity exerted by FGF2 in cultured endothelial cells. Finally, it inhibited angiogenesis in a chick embrio chorioallantoic membrane (CAM) assay in which heparin is inactive.