One of the proposed pathways to the synthesis of 4-hydroxy-nonenal (HNE) and related aldehydes entails formation of an intermediate bis-allylic fatty acid dihydroperoxide. As a first direct demonstration of such a pathway and proof of principle, herein we show that 8R-lipoxygenase (8R-LOX) catalyzes the enzymatic production of the HNE-like product (11-oxo-8-hydroperoxy-undeca-5,9-dienoic acid) via synthesis of 8,11-dihydroperoxy-eicosa-5,9,12,14-tetraenoic acid intermediate. Incubation of arachidonic acid with 8R-LOX formed initially 8R-hydroperoxy-eicosatetraenoic acid (8R-HPETE), which was further converted to a mixture of products including a prominent HPNE-like enone. A new bis-allylic dihydroperoxide was trapped when the incubation was repeated on ice. Reincubation of this intermediate with 8R-LOX successfully demonstrated its conversion to the enone products, and this reaction was greatly accelerated by coincubation with NDGA, a reductant of the LOX iron. These findings identify a plausible mechanism that could contribute to the production of 4-hydroxy-alkenals in vivo.