Fibroblast growth factor-2 (FGF-2), a potent angiogenic factor, requires heparin for dimerization and activation of the FGF receptor tyrosine kinase. The binding of multiple fibroblast growth factors by heparin may be necessary for dimerization of the FGF receptor. Analytical ultracentrifugation of FGF- 2 in the presence of heparin-derived saccharides shows that both an active heparin octasaccharide and an inactive heparin-like disaccharide induce fibroblast growth factor-2 self-association. Analysis of the data indicates that the heparin octasaccharide induces a monomer-dimer-tetramer assembly of FGF-2 while the disaccharide induces a monomer-dimer equilibrium. Evidence is presented indicating that the dimer conformation induced by the heparin octasaccharide is a side by side dimer with the FGF-2 molecules cis to the heparin, while the disaccharide-induced dimer is a head to head dimer in which FGF-2 molecules are trans to the ligand. These results, combined with previous studies, support the model that formation of a specific side by side heparin-induced FGF-2 dimer is required for activation of the FGF receptor.