Mammalian lipoxygenases are implicated in the biosynthesis of inflammatory mediators, in the pathogenesis of atherosclerosis and in the process of blood cell differentiation and maturation. With respect to their reaction specificity, three major types of mammalian lipoxygenases (15-lipoxygenases, 12-lipoxygenases and 5-lipoxygenases) may be classified. Although this nomenclature is commonly used, the mechanistic reasons for the positional specificity of lipoxygenases are not well understood. We investigated the structural reasons for lipoxygenase specificity by a combination of chimera formation and site-directed mutagenesis, and identified phenylalanine 353 as primary determinant for the positional specificity of rabbit reticulocyte 15-lipoxygenase. Modeling of the enzyme-substrate interaction suggested that the alignment of arachidonic acid at the active site appears to be influenced by this residue. According to the substrate orientation, the 15-lipoxygenase may be differentiated from two types of mammalian 12-lipoxygenases.