The urea-induced unfolding of acidic fibroblast growth factor (aFGF) in the presence and absence of various polyanions has been quantitatively examined by fluorescence spectroscopy. In the absence of a stabilizing polyanion, the apparent free energy of unfolding of aFGF is 6.5 kcal mol-1. The presence of equimolar or greater amounts of heparin stabilizes aFGF from unfolding by more than 2.5 kcal mol-1 and slows the rate of unfolding by greater than 2000-fold. The ability of heparin to stabilize aFGF is critically dependent upon many factors including the number of aFGF molecules bound to the heparin chain, ionic strength, temperature, and the extent of sulfation of the polysaccharide. The presence of similar amounts of other polyanions such as sulfated beta-cyclodextrin or heparan sulfate also stabilizes aFGF to a similar extent as heparin. Additional experiments demonstrate that increasing charge density enhances the ability of polyanions such as sulfated beta-cyclodextrins, phosphorylated inositols, and modified heparins to protect aFGF from urea-induced unfolding.