The mitogenic activity of acidic fibroblast growth factor (aFGF) is potentiated by the highly sulfated hexasaccharide [IdoUA,2S-GlcNS,6S]2-[GlcUA-GlcNS,6S] the structural repetitive unit of lung heparin chains. On a mass basis, the effect of both heparin and oligosaccharide are equivalent whereas on a molar basis, heparin, which contains about seven hexasaccharide repeats, is more efficient. On the other hand, a pentasulfated tetrasaccharide or di- and tri-sulfated disaccharides are much less effective in potentiating aFGF activity than the hexasaccharide. If the growth factor is pre-incubated with the hexasaccharide at pH 7.2 and then exposed to pH 3.5 the 306/345 nm fluorescence ratio is similar to that of native aFGF indicating that the oligosaccharide stabilizes a native conformation of the protein. Heparan sulfates extracted from various mammalian tissues were also able to potentiate aFGF mitogenic activity. On a mass basis they were in general less efficient than heparin; however, heparan sulfate prepared from medium conditioned by 3T3 fibroblasts is more efficient than heparin both on a mass and molar basis. A highly sulfated oligosaccharide isolated after digestion of pancreas heparan sulfate with heparitinase I is more active than the intact molecule, reaching a potentiating effect equivalent to that of lung heparin, whereas an N-acetylated oligosaccharide isolated after nitrous acid degradation is inactive. These data suggest that the mitogenic activity of aFGF is primarily potentiated by interacting with highly sulfated regions of heparan sulfates chains.