Acetone dried human pituitary powder was preextracted for gonadotropins according to the method of Hartree (1). Two components of human neurophysin, designated H-Np I and H-Np II, were purified from the 75% ethanol supernatant which remains after precipitation of the gonadotropins. The neurophysin in the supernatant was precipitated by the addition of two volumes of acetone. The precipitate was redissolved and fractionated by gel nitration on Sephadex G-100. The two components of neurophysin were resolved by DEAE-cellulose chromatography and purified further by gel filtration on Sephadex G-75. H-Np I was one of the major protein components in the human posterior pituitary lobe and was also the principal neurophysin identified. The molecular weights of H-Np I and H-Np II were estimated to be 10,000. The amino acid composition of these polypeptides was similar to that of bovine and porcine neurophysins, being rich in 1/2 cystine, containing only one residue of tyrosine and no tryptophan residues, but one residue of histidine. H-Np II is differentiated from H-Np I by having one residue of methionine per mole of protein. The N-terminal amino acid residue of both is alanine. The electrophoretic mobility of H-Np I upon starch gel electrophoresis at pH 8.1 is more rapid than that of H-Np II; both components have the characteristic ultraviolet absorption spectrum of neurophysin; that is no defined absorption maximum at 275–280 mu. Peptide maps of trypsin-digests of human, bovine, and porcine neurophysin revealed nine peptide fragments. Six of these peptides from all three species moved to similar positions after paper electrophoresis and chromatography.