Production of radiolabeled arachidonic acid (AA) metabolites by microsomal fractions obtained from rabbit renal papillae and cortices was investigated. It was determined that in addition to the cyclooxygenase products synthesized, the papillary fractions and the cortical fractions each produced 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE), 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) and three leukotrienes (LT). The LT of greatest polarity showed an ultraviolet (UV) spectrum of a conjugated triene chromophore and coeluted with standard LT C4 on reverse phase high performance liquid chromatography (RP-HPLC). Amino acid analysis revealed the presence of a glutathionyl moiety, whereas mass spectrometric analysis of the trimethylsilyl ether and methyl ester derivative of this LT indicated the lipid portion to be 5-hydroxy-arachidic acid. In addition, this LT was found to contract guinea pig ileum. The other LTs were found to lack bioactivity on guinea pig ileum but contained a glutathionyl moiety. These findings indicate that rabbit renal microsomes possess lipoxygenase activity capable of producing hydroxy acids, such as 5-HETE, 12-HETE and LT.