Tryptophan 5-monooxygenase was purified 880-fold with a 48% yield from mouse mastocytoma cells (P815) by only a one-step purification procedure of pteridine affinity chromatography. The specific activity of the final preparation was 5280 nmol min-1 mg-1. It gave a single protein band on polyacrylamide gel electrophoresis in the absence and presence of sodium dodecylsulfate. The molecular weight of the enzyme was determined to be 270,000 by gel filtration and 280,000 by gradient polyacrylamide gel electrophoresis. Sodium dodecylsulfate/polyacrylamide gel electrophoresis revealed the enzyme to be composed of identical subunits with a molecular weight of 53,000. Tetrameric structure of the enzyme was suggested by cross-linking studies using dimethyl suberimidate as a bifunctional reagent. The isoelectric point of the enzyme was estimated to be 6.0. Amino acid analysis showed a residue composition similar to that reported for rat liver phenylalanine 4-monooxygenase. The enzyme activity was stimulated approximately fivefold by preincubation with dithiothreitol and Fe2+. The purified enzyme had an activity of phenylalanine hydroxylation and also a weak activity of tyrosine hydroxylation. The kinetic properties of the enzyme are also presented.