A single protein from human leukocytes possesses both 5-lipoxygenase and leukotriene A4 (LTA4) synthase activities. It has been reported that LTA4 production is more efficient when the enzyme utilizes arachidonic acid, than when 5-HPETE is exogenously supplied as substrate. In the present study, human leukocyte homogenate 100,000 X g supernatant was incubated with 100 microM octadeuterated arachidonic acid and exogenous 5-HPETE (0-80 microM), and the isotopic composition of LTA4 hydrolysis products was determined by gas chromatography-mass spectrometry. Even though 100 microM deuterated arachidonic acid results in 20-30 microM deuterated 5-HPETE, 80 microM exogenous 5-HPETE in the incubation could reduce the amount of deuterated LTA4 by only approx. 20%. The present study would thus indicate that the arachidonic acid moiety is preferentially converted to LTA4 in a concerted reaction without dissociation of a 5-HPETE intermediate.