[en] In order to identify regulatory steps in leukotriene synthesis, the biochemical characteristics of a 5-lipoxygenase activity in a murine mast cell clone (MC-9) were investigated. Supernatants (100,000xg) of sonicated cells metabolized ¹⁴C-arachidonic acid to leukotriene B4 (LTB₄), diastereiomeric 5,12-dihydroxy-eicosatetraenoic acids (5,12-diHETEs), 5-hydroperoxy- and 5-hydroxy-eicosatetraenoic acids (5-HPETE and 5-HETE) and 5-oxo-eicosatetraenoic acid which were identified by high performance liquid chromatography (HPLC) and gas chromatograph-mass spectrometry (GC/MS). Lipoxygenase activity had a pH optimum of 6.9 and was highly dependent upon added calcium. The concentration of calcium for 50% activation (EC₅₀) was 3μM. Activity was also stimulated by ATP (EC₅₀ = 160 μM). Lipoxygenase activity exhibited a biphasic concentration dependence for arachidonic acid with maximum product formation occurring at 35 μM. The activity showed apparent lag phase kinetics which were more pronounced at low protein levels (0.8 mg/ml). The lag phase was also greatly accentuated by glutathione (1 mM) plus glutathione peroxidase (0.4 units/ml). In contrast, addition of any of several hydroperoxides, i.e. 5-, 8-, 9-, or 15-HPETEs (EC₅₀ 1 μM), shortened the lag phase. The results suggest that the cellular levels of hydroperoxides and glutathione peroxidase, as well as calcium and certain nucleotides, may be important factors regulating leukotriene synthesis