Tryptophan hydroxylase from rat mesencephalic tegmentum has been purified by sequential chromatography on Blue-Sepharose, DEAE, and calmodulin-Sepharose. The hydroxylase is excluded from Blue-Sepharose and is eluted from DEAE with a stepwise NACl gradient. Finally, tryptophan hydroxylase binds to calmodulin-sepharose and is eluted with EGTA. The purification scheme is rapid and yields an enzyme with a specific activity of 3.5 nmol 5HTP/mg min, representing a 61-fold purification with 8% recovery.