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We tested a simple model which explains the singular or dual specificity of lipoxygenases. The dual specificity considered here is typified by the oxygenation of arachidonic acid by the reticulocyte lipoxygenase: two chiral products are formed (12S- and 15S-hydroperoxides, ratio approximately 1:9) via hydrogen abstraction from two separate methylene groups (C-10 and C-13). The rate-limiting step is known to involve this hydrogen abstraction, and we assumed that alignment of the methylenes with the hydrogen acceptor on the enzyme is critical in terms of reaction rate and positional specificity. Optimal alignment will be associated with a fast rate of reaction and formation of a single chiral product. A shift in position of the double bonds (and hence of the methylene groups) should be associated with a slower rate of reaction and formation of two chiral products; two methylenes are now able to react, although neither has perfect alignment. We tested this idea using two lipoxygenases and polyenoic fatty acids differing in the number and position of the double bonds. Optimal substrates for the soybean lipoxygenase had a doubly allylic methylene in the n-8 position, while the reticulocyte enzyme preferred substrates with a n-9 methylene. These substrates were converted to a single chiral product. With both enzymes, the other series of substrates reacted more slowly and were converted to two chiral products. We conclude that alignment of methylene groups of the substrate at the active site is a major determinant of the reaction rate and the singular or dual specificity of lipoxygenases.