The structural properties of fibroblast heparan sulfate (HS) that are necessary for it to bind strongly to basic fibroblast growth factor (bFGF) have been investigated using bFGF affinity chromatography. Specific enzymic and chemical scission of HS, together with chemical N-desulfation, revealed that N-sulfate groups and iduronate-2-sulfates (IdoA(2-OSO3)) were essential for the interaction. bFGF-affinity chromatography of sulfated oligosaccharides released from HS by treatment with heparitinase led to the identification of an oligosaccharide component (oligo-H), seven disaccharides in length, with a similar affinity for bFGF as the parent molecule. Heparinase treatment of this fraction abolished the high affinity binding to bFGF. Analysis of oligo-H indicated that 74% of the disaccharide units had the structure IdoA(2-OSO3)alpha 1,4GlcNSO3; the remainder comprised N-acetylated and N-sulfated units, the majority of which were devoid of O-sulfate groups. Oligo-H was fully degraded to disaccharides by treatment with nitrous acid. These results indicate that the sequence of oligo-H is as shown below. delta GlcA beta 1,4GlcNSO3 alpha 1,4[IdoA(2-OSO3)alpha 1,4GlcNSO3]5 alpha 1, 4IdoA alpha 1,4GlcNAc Sulfated oligosaccharides of similar size but with a lower affinity for bFGF had a reduced concentration of IdoA(2-OSO3) but significant quantities of GlcNSO3(6-OSO3) and GlcNAc(6-OSO3). The data indicate a primary role for contiguous sequences of IdoA(2-OSO3)alpha 1,4GlcNSO3 in mediating the high affinity binding between fibroblast HS and bFGF.