This study aimed to check the involvement of lipid mediator leukotriene (LT) B4 and the activity of LTA4 hydrolase (LTA4H) in the development of arthritis induced in rats by collagen and adjuvant (CIA). High-performance liquid chromatography (HPLC) and enzyme immunoassay (EIA) were used for measurements of LTB4 and LTA4H in plasma, synovial fluid (SF), soluble (SO), and solubilized membrane-bound fraction (MB) from synovial tissue (ST) and peripheral blood mononuclear cells (PBMCs) of CIA-arthritic and CIA-resistant. EIA process is simple, clean, and rapid and offered advantages over HPLC, showing that in SF and MB-PBMCs of CIA-arthritic and CIA-resistant, and in MB-ST of CIA-resistant, LTB4 and LTA4H were altered in parallel and were positively related. In the plasma and SO-ST and SO-PBMCs of CIA-arthritic and CIA-resistant, and in MB-ST of CIA-arthritic, this pattern was not found. The primordial role played by LTA4H in the biosynthesis of LTB4 was confirmed together with the existence of alternative steps that regulate LTB4 without participation of LTA4H. The involvement of compartmentalized and coupled changes of LTB4 and LTA4H in the resistance and development of arthritis in CIA model was demonstrated for the first time.