The eggshell matrix was obtained from hen eggshells using EDTA solutions. The water-soluble organic material was separated on a DEAE-cellulose column equilibrated with 8 M urea using Tris-hydrochloric acid buffer as eluent with a linear gradient of sodium chloride. Five main fractions were obtained which differ in amino acid composition and sugar contents. As is shown from the uronic acid content, the first two fractions eluted from the column are glycoproteins, while the other three contain proteins and glycosaminoglycans From the alkaline hydrolysate of the eggshell matrix, a peptide was isolated which is composed of aspartic acid, threonine, serine, glutamic acid, proline, glycine and alanine in a molar ratio of 2:1:3:7:1:3:1 with a minimum molecular weight of 2158 daltons. The calcium ion binding to this peptide was studied, at different pH values, with both free and blocked carboxyl groups, using murexide as an indicator of free Ca2+. The importance of this acidic peptide in the calcification process of the eggshell matrix is discussed.