The kinetics of the transformation of arachidonic acid into 5-lipoxygenase products in human blood leukocytes stimulated with the ionophore A23187 (2 microM) was studied using high performance liquid chromatography. The levels of 5-hydroxyeicosatetraenoic acid (5-HETE), leukotriene B4 (LTB4), delta 6-trans-LTB4, and leukotriene C4 (LTC4) were maximum after 4-5 min of incubation; at longer incubation times, the levels of delta 6-trans-LTB4 and LTC4 remained unchanged, whereas those of 5-HETE and LTB4 decreased rapidly. The disappearance of LTB4 was concomitant with the formation of omega-hydroxy-LTB4 and omega-carboxy-LTB4. When synthetic LTB4 was added to a suspension of unstimulated human leukocytes, a similar pattern of omega-oxidation products was observed. This omega-oxidation process showed some specificity for LTB4, as indicated by the slower reaction of three stereoisomers of LTB4. Studies with subpopulations of human blood cells and human plasma clearly indicated that the polymorphonuclear leukocytes were the main source of enzymic activity for the omega-oxidation of LTB4. The liquid chromatographic behaviors of natural omega-hydroxy-LTB4 and omega-carboxy-LTB4 were studied in two systems.