5-Lipoxygenase (5-LO) is a key enzyme involved in leukotriene (LTs) biosynthesis which act as host defense mediators, and inflammatory agents as well. In this work the influence of anionic cholesterol derivatives on 5-LO activity has been investigated. Cholesterol sulfate activated human polymorphonuclear leukocytes (PMNL) and stimulated their adhesion to endothelium and collagen. Cholesterol sulfate and cholesterol phosphate suppressed leukotriene production in PMNL and in rat RBL-1 cells. Kinetic characteristics of this process are presented. Cholesterol phosphate (charge -2) was shown to be more potent inhibitor then cholesterol sulfate (charge-1) in all experiments. We suppose that this fact highlights the importance of negatively charged ester groups to suppress 5-LO activity.