Leukotriene A4 synthase was purified from the cytosolic fraction of murine mast cells. The enzyme converted 5-hydroperoxy-6-trans-8,11,14-cis-icosatetraenoic acid (5-HPETE) to leukotriene A4. This unstable product was identified by demonstration of two epimers of 6-transleukotriene B4, methanol trapping, as well as further transformation to leukotriene B4 by leukotriene A4 hydrolase. Leukotriene A4 synthase stereospecifically eliminated the D-hydrogen at C-10 (pro-R) in the synthesis of leukotriene A4 when incubated with [10D-3H;3-(14)C]5-HPETE. The purified enzyme also exhibited 5-lipoxygenase activity toward arachidonic acid and 8-lipoxygenase activity towards 8,11,14-cisicosatrienoic acid. All of these activities required Ca2+ and ATP for their maximal velocities. The effects of heat treatment and of several lipoxygenase inhibitors on these enzyme activities as well as coelution in various chromatographic systems strongly suggest that lipoxygenase and leukotriene A4 synthase activities reside in the same enzyme molecule.