Lipoxin B4 (LXB4) is metabolized either by human neutrophils or by the neutrophil microsomes to a polar compound on a reverse-phase high-performance liquid chromatography. The metabolite is identified as 20-hydroxy-lipoxin B4 (20-OH-LXB4), a novel member in the arachidonic acid cascade, on the basis of ultraviolet spectrometry and gas chromatography-mass spectrometry. The neutrophil microsomes convert LXB4 to its 20-hydroxy derivative under aerobic condition in the presence of NADPH. The reaction is inhibited by carbon monoxide, an inhibitor of cytochrome P-450 (P-450), and by antibodies raised against NADPH-P-450 reductase. A P-450 is thus involved in the omega-hydroxylation of LXB4. The P-450 appears to be the one responsible for leukotriene B4 (LTB4) omega-hydroxylation, P-450LTB omega, based on the following observations. The formation of 20-OH-LXB4 is inhibited solely by substrates of P-450LTB omega such as LTB4 and leukotriene B5 among various fatty acids including prostaglandins. The order of the inhibitory potencies of these substances on the LXB4 omega-hydroxylation is the same as that of their affinities for LTB4 omega-hydroxylase. LTB4 inhibits the reaction in a competitive manner with the Ki value of 0.2 microM, which agrees with the Km value for the LTB4 omega-hydroxylation (0.3 microM).