Analysis by gas chromatography/mass spectrometry (GC/MS) of derivatized metabolites formed following incubation of leukotriene B, (LTB,) incubation with Ito cells extends previous knowledge concerning fragmentation mechanisms for derivatized hydroxy-substituted unsaturated fatty acids. LTB, was metabolized by rat Ito fells, a hepatic perisinusoidal stellate cell, by the 6' ' -and A"-reductase pathways, resulting in the formation of 10,lldihydro-LTB, and 10,11,14,15-tetrahydro-LTB4. Formation of the intermediate metabolites, 12-0x0-10,11dihydro-LTB, and 12-ox0-l0,11,14,15-tetrahydro-LTB4, was also observed. GC/electron impact (EI) MS analysis of the 12-ox0 metabolites, derivatized as the pentafhorobenzyl ester/trimethylsilyl ether compounds, resulted in unique fragmentations indicative of the 0x0 substituent and double bond positions. Further metabolism of 10,lldihydro-LTB, and 10,11,14,15-tetrahydro-LTB4 by carboxy terminus B-oxidation resulted in chain-shortened monohydroxy metabolites. Possible intermediates in this metabolism, which resulted in loss of the original C-5 hydroxy substituent from LTB, , were identified as 2,4,6-conjugated triene-containing C-18 metabolites. The absence of a double bond allylic to the trimethylsiloxy ether in derivatized 10,11,14,15-tetrahydro LTB, metabolites strikingly reduced the abundance of a-cleavage ions observed in the EI mass spectra of these compounds, thus suggesting the importance of formation of an allylic stabilized radical in such a-cleavage reactions. Lacking a favorable a-cleavage reaction, GC/EIMS analysis of 1O-hydroxy-2,4,6-octadecatrienoic acid resulted in the formation of m/z 91, which may arise via cyclizatiou of the conjugated triene moiety. In addition, GC/MS analysis of derivatized metabolites containing the 2,4,6 conjugated triene moiety resulted in a unique fragment ion in the electron capture ionization mass spectra that also may arise via cyclization of the conjugated triene with formation of mlz 121.