Arachidonate 5-lipoxygenase was purified 400-fold from homogenates of human peripheral blood leukocytes by a combination of ammonium sulfate fractionation, gel filtration chromatography, and HPLC on anion exchange and hydroxylapatite columns. NaDodSO4/polyacrylamide gel electrophoresis of the purified protein revealed the presence of a single major band (apparent Mr, 80,000). Densitometric analysis of the Coomassie blue staining pattern of the gels revealed that a 90-97% purity had been achieved. As has been reported for the 5-lipoxygenase from other mammalian sources, the human leukocyte enzyme required Ca2+ and ATP for maximal activity. In addition, a number of factors were isolated during the course of the purification, which possessed significant 5-lipoxygenase stimulatory activities. These were obtained in a high-speed pellet of leukocyte homogenate, a 60-90% ammonium sulfate precipitate fraction, and the unabsorbed protein from the first anion exchange HPLC step. In the absence of stimulatory factors, little activity was detected in the purified enzyme, even in the presence of Ca2+ and ATP. The specific function of these various factors is unknown, but their existence suggests that the human leukocyte 5-lipoxygenase is regulated by a complex mechanism that is likely to play an important role in the control of leukotriene and lipoxin biosynthesis.