Bovine neurophysin II (BNP‐II), a major neurohypophyseal hormone‐binding protein in the cow, was isolated from acetone‐dried posterior pituitary powder by gel filtration, ion exchange chromatography and preparative disc electrophoresis. The single‐chain protein is composed of 97 amino acids, possesses a molecular weight of 10,029, and contains seven disulfide bonds. The fully S‐alkylated protein, S‐carboxamido‐[C]‐ methylcysteine BNP‐II ([C]BNP‐II), was subjected to 80 automated cycles of the Edman degradation with positive identification of every amino acid residue through 65 cycles and many identifications through 80 residues. [C]BNP‐II was digested with chymotrypsin and the peptides were isolated by gel filtration and purified by ion exchange chromatography. A 55‐amino acid C‐terminal chymotryptic peptide was sequenced through 46 residues by automated Edman degradations producing a 38‐amino acid overlap with the sequence of [C]BNP‐II. This established the sequences of the first 88 residues of the protein. A chymotryptic nonapeptide was sequenced from NH2‐ to COOH‐terminus by manual Edman degradations which established the sequence through residue 94. COOH‐terminal analysis of aminoethylated BNP‐II elucidated the tetrapeptide sequence and produced an overlap with the sequenced chymotryptic nonapeptide, completing the proposed amino acid sequence, which is supported by amino acid compositions of three other