Crystalline human insulin was prepared from human pancreas glands in relatively large amounts employing acid-ethanol extraction, fractional precipitation with salts and organic solvents, gel filtration, and crystallization from ammonium acetate buffer. The crystalline insulin had a potency of about 25 International Units per milligram. Polyacrylamide gel electrophoresis showed that the material consisted of a major component and several minor components similar to those usually observed in insulins derived from other species. Amino acid analyses of the crystalline material were consistent with the structure proposed for human insulin by Nicol and Smith.