A recombinant human basic fibroblast growth factor CS23 mutein (rhbFGF-CS23) obtained from Escherichia coli cells has proliferation-stimulating activity for a fetal bovine heart endothelial cell line, ATCC CRL 1395 (biological activity), and strong affinity for heparin (heparin-affinity) similarly to the natural human basic fibroblast growth factor. Plural species having different kinds of heparin-affinity were formed by acetylation of rhbFGF-CS23 with acetic anhydride. To clarify the relationship between the sites with heparin-affinity and the biologically active sites, we have investigated the acetylation sites by peptide mapping and the biological activity of the acetylated species. Consequently, the sites with heparin-affinity in rhbFGF-CS23 are found to be Lys²⁶, Lys¹¹⁹, Lys¹²⁵, Lys¹²⁹, and Lys¹³⁵, in the primary structure, and these sites with heparin-affinity except Lys²⁶ are also clarified to be very important to retain the biological activity of the factor.