Six previously unidentified leukotriene (LT) B4 metabolites formed during incubation of LTB4 with human keratinocytes in primary culture indicate the importance of the 12-hydroxyeicosanoid dehydrogenase pathway in LTB4 metabolism. The ultraviolet absorption spectra obtained for all keratinocyte metabolites revealed the presence of a conjugated diene structural moiety rather than the conjugated triene structure of LTB4. Metabolites were characterized using fast atom bombardment-mass spectrometry, gas chromatography-mass spectrometry of the pentafluorobenzyl ester, trimethylsilyl ether derivatives and specific degradation reactions. The previously identified 10,11-dihydro-LTB4 and 10,11-dihydro-12-epi-LTB4 were observed as well as 20-OH-10,11-dihydro-LTB4, consistent with the reductase pathway of LTB4 metabolism. This pathway involves initial formation of 12-oxo-LTB4 catalyzed by 12-hydroxyeicosanoid dehydrogenase followed by reduction by delta 10-reductase. The most lipophilic metabolite of LTB4 was identified as 10-hydroxy-4,6,12-octadecatrienoic acid which could result from beta-oxidation reactions of LTB4 following reduction of the 10,11-double bond. One of the most abundant metabolites was characterized as 3,7,14-trihydroxy-8,10,16- docosatrienoic acid which could result from fatty acid elongation following reduction of the 10,11-double bond. Additional abundant LTB4 metabolites were identified that result from glutathione conjugation of 12-oxo-LTB4. These were characterized using fast atom bombardment-mass spectrometry and by chemical degradation using hypochlorous acid as well as transpeptidases. These metabolites were identified as 5,12-dihydroxy-6-glutathionyl-7,9,14-eicosatrienoic acid (c-LTB3), 5,12-dihydroxy-6-cysteinyl-glycyl-7,9,14-eicosatrienoic acid (d-LTB3) and 5,12-dihydroxy-6-cysteinyl-7,9,14-eicosatrienoic acid (e-LTB3). We propose that these metabolites result from a 1,8 Michael-type addition of glutathione to the 12-oxo-LTB4 intermediate.