Leukotriene B4 (LTB4) 12-hydroxydehydrogenase was purified to apparent homogeneity from the cytosol fraction of the porcine kidney. The N-terminal amino acid sequence analysis revealed that this enzyme is a novel protein with a molecular weight of 35,000. Although the enzyme is ubiquitously distributed in various tissues and leukocytes of porcine, the kidney and liver had the highest enzyme activities. In the presence of NADP+ as a cofactor, the enzyme catalyzes the conversion of LTB4 to 12-oxo-LTB4, the structure identified by gas chromatography/mass spectrometry. 12-Oxo-LTB4 was further converted by other enzymes to 10,11,14,15-tetrahydro-12-oxo-LTB4, which was determined by proton NMR and gas chromatography/mass spectrometry. 12-Oxo-LTB4 was 100-fold less potent than LTB4 in increasing intracellular calcium concentrations of human leukocytes. 6-trans-LTB4 and LTB4 proved to be the best substrates of the enzyme, whereas various types of monohydroxyeicosatetraenoic acids, 5(S),12(S)-dihydroxyeicosatetraenoic acid, prostaglandins, cortisol, or pregnenolone could not serve as a substrate. These results suggest that the enzyme acts specifically on the 12(R)-hydroxy group of leukotriene B4 and is involved in the metabolic inactivation of LTB4 in the porcine kidney.