FGF-1 binds to and activates specific transmembrane receptors (FGFRs) and is subsequently internalized and translocated to the interior of the cell. To elucidate the role of the receptor in the translocation process, we studied the effects of the elimination of distinct sites of the ligand-receptor interaction. On the basis of the structure of the FGF-1-FGFR1 complex, we substituted four key amino acid residues of FGF-1 from the FGF-receptor binding site with alanines, constructing four point mutants and one double mutant. We determined by in vivo assays in NIH 3T3 cells the ability of the mutants to bind to specific FGF receptors, to stimulate DNA synthesis, and to activate downstream signaling pathways. We found that correct binding to the receptor is necessary for optimal stimulation of DNA synthesis. All four single mutants became phosphorylated to different extents, indicating that they were translocated to the cytosol/nucleus with varying efficiency. This indicates that despite a low affinity for FGFR, translocation to the cytosol/nucleus can still occur. However, simultaneous substitution in two of the positions led to a total loss of biological activity of the growth factor and prevented its internalization, implying that there is only one strongly receptor-dependent, productive way of translocating FGF-1. We also found that the process of translocation did not correlate with the thermal stability of the protein. Additionally, we observed a clear negative correlation between the stability of the FGF-1 mutants and the efficiency of their phosphorylation, which strongly suggests that protein kinases prefer the unfolded state of the protein substrate.