The present studies were conducted to characterize the specific binding of recombinant human [125I]acidic fibroblast growth factor (aFGF) to the cloned human fibroblast growth factor (FGF) receptor, flg, overexpressed on stably transfected NIH 3T3 mouse fibroblast (NFlg26) cell membranes. In the presence of 5 U/ml of heparin to block [125I]aFGF binding to membrane bound heparan sulfate proteoglycans, specific [125I]aFGF binding was optimal in the presence of 0.2 M NaCl and in a pH range of 7 to 9. [125I]aFGF labeled a single class of recognition sites with high affinity (Kd = 0.27 nM) and limited capacity (apparent maximum binding = 19.5 pmol/mg of protein). A similar estimate of ligand affinity (Kd = 0.25 nM) was determined from association and dissociation rate experiments. aFGF, basic fibroblast growth factor and several glycine-substituted point mutations of aFGF potently inhibited 0.1 nM [125I]aFGF binding. A variety of putative FGF receptor ligands including poly-L-lysines and poly-L-arginines, protamine, suramin and wheat germ agglutinin were shown to have weak or no affinity for the [125I]aFGF recognition site. Additional saturation studies, conducted in the presence of a lower (0.1 U/ml) heparin concentration, indicated that [125I] aFGF labeled both the high affinity (Kd = 0.02 nM) FGF-flg receptor and a separate class of lower affinity (Kd = 2 nM) recognition sites. Pretreatment of NFlg26 cell membranes with pertussis toxin resulted in a heparin-dependent decrease in the binding affinity (Kd values of 0.57-1.15 nM) of [125I]aFGF. Similar pretreatment with cholera toxin did not significantly affect [125I] aFGF binding. Guanine nucleotides were also found to significantly reduce 0.1 nM [125I]aFGF binding in a heparin-dependent fashion. The present data demonstrate that, in the presence of heparin, [125I]aFGF binds with high affinity to the cloned FGF-flg receptor on NFlg26 cell membranes. However, at a low heparin concentration (0.1 U/ml), [125I]aFGF binds to the FGF-flg receptor with higher affinity than was observed in the presence of 5 U/ml of heparin, and also binds a class of lower affinity recognition sites which are consistent with the labeling of cell surface heparan sulfate proteoglycans. The present data also indicate that agents which are known to interfere with receptor/G-protein coupling reduce the binding affinity of [125I]aFGF and suggest that the FGF-flg receptor may be coupled to a G-protein in addition to its intrinsic tyrosine kinase activity.