Mitochondrial membranes and plasma membranes of rabbit reticulocytes contain oxygenated polyenoic fatty acids such as (9Z,11E)-(13S)-13-hydroxy-9,11-octadecadienoic acid, 9S and 9R isomers of (10E,12Z)-9-hydroxy-10,12-octadecadienoic acid and their all-E isomers. Furthermore (5Z,8Z,11Z,13E)-(15S)-15-hydroxy-5,8,11,13-icosa tetraenoic acid, 9- and 13-oxooctadecadienoic acid were detected as minor products. The chemical structure of these products has been identified by co-chromatography with authentic standards, by ultraviolet and infrared spectroscopy, and by gas chromatography/mass spectrometry of the native compounds and their hydrogenated derivatives. The oxygenated fatty acids originate most probably from the intracellular action of the erythroid arachidonate 15-lipoxygenase. In membranes of the mature erythrocyte only small amounts of hydroxy fatty acids were detected. Young peripheral reticulocytes contain more oxygenated polyenoic fatty acids in their membranes than older cells. In mixed cell populations, about 85% of the lipoxygenase products were found esterified to the membrane ester lipids, whereas 15% were associated as free hydroxy fatty acids with the membranes. The hydroxy fatty acid content of the mitochondrial membranes is more than threefold higher than that of the plasma membranes. The pattern of the products isolated from plasma membranes shows a high specificity with (9Z,11E)-(13S)-13-hydroxy-9,11-octadecadienoic acid as the main product. In contrast, the pattern found in the mitochondrial membranes was much more unspecific: a complex mixture of all positional and optical isomers was detected. The data presented indicate that the reticulocyte lipoxygenase in vivo acts on both plasma membranes and mitochondrial membranes. The results are discussed in the light of the involvement of the lipoxygenase in the breakdown of mitochondria and other organelles in reticulocytes during maturation.