The structure of human placental calphobindin-II (CPB-II) was investigated by amino acid composition and amino acid sequence analyses of peptides generated by protease digestion of the protein. The 45 peptides obtained from the lysyl endopeptidase digest of CPB-II, and the amino-terminal peptide prepared from its tryptic digest, were analyzed, and they accounted for over 98% of total amino acids of CPB-II. The structure of CPB-II determined by protein sequencing was identical to that previously predicted from its cDNA sequence (Iwasaki, A. et al. (1989) J. Biochem. 106, 43-49), except for the amino terminus. Since the amino terminus of CPB-II was blocked to Edman degradation, fast-atom-bombardment mass spectrometric analysis was used to demonstrate that the amino-terminal residue was acetyl-alanine. The carboxyl-terminal residue of CPB-II was identified as aspartic acid by the hydrazinolytic procedure. Calcium-binding studies indicated that 1 mol of CPB II binds 1 mol of calcium in the absence of phospholipid and 8 mol of calcium in the presence of phospholipid.