We recently demonstrated that proteoglycan macrophage colony-stimulating factor (PG-M-CSF) binds basic fibroblast growth factor (bFGF) and neutralizes the biological activity of bFGF. In this study, we identified the binding sites of PG-M-CSF for bFGF. We examined the binding of bFGF to overlapping 12-mer peptides with the sequence of the putative binding region. High affinity binding was detected at two peaks; one consisted of the three adjacent peptides, 212-223, 213-224 and 214-225 and the other, of the three adjacent peptides, 246-257, 247-258 and 248-259. The synthetic peptide (212VDPGSAKQRPPRST225) did not inhibit bFGF binding to another peptide (246PQPRPSVGAFNPGM259), and vice versa. However, both peptides inhibited the bFGF-induced but not platelet-derived growth factor-induced stimulation of DNA synthesis in murine Balb/c 3T3 cells.