There ate four EF-hand polypeptides in calmodulin, a natural ubiquitous calcium binding protein that activates the enzymes involved in Ca2+-mediated signal transduction. An EF-hand polypeptide has six carboxylate functional groups in the middle loop region between two rigid polypeptides. In this study, a calcium binding polymer (CBP) with a structure of poly(L-alanine)-poly(L-alanine-co-L-glutamic acid)-poly(ethylene glycol)-poly(L-alanine-co-L-glutamic acid)-poly(L-alanine) (PA-PAE-PEG-PAE-PA; A(11.1)-A(3.4)E(3.2)-EG(40.1)-A(3.4)E(3.2)-A(11.1)) was synthesized by mimicking the EF-hand polypeptide. The 6-7 carboxylate functional groups from PAE are expected to form a binding site for Ca2+. As the Ca2+ bound to CBP, small changes in the circular dichroism spectra and C-13 NMR spectra were observed, indicating that Ca2+ binding to CBP induced changes in the conformation of CBP. The binding constant of CBP to Ca2+ was investigated by using the competitive binding of 2,2',2 '',2'''-{ethane-1,2,diylbis[oxy(4-bromo-2,1-phenylene)nitrilo]} tetraacetie acid (5,5-Br-2-BAPTA). The binding constant obtained with a CaLigator program by least-squares fitting of the absorbance profile as a function of Ca2+ concentration was 5.1 X 10(5) M-1, which was similar to that of calmodulin. The selectivity of CBP for metal ion binding was compared among Ca2+, Cu2+, and Zn2+. The binding constant was obtained through. a similar competitive binding study with murexide. The binding constants for Ca2+, Cu2+, and Zn2+ were 7.0 X 10(5), 4.2 X 10(5), and 1.7 X 10(5) respectively, indicating 2-4 fold higher selectivity of CBP for Ca2+ compared to Cu2+ and Zn2+. The,CBP has selectivity for Ca2+, and binding affinity for Ca2+ was similar to the biological Ca2+ binding motif of calmodulin.