A lipoxygenase has been found in the reticulocytes of all mammalian species tested so far (rabbit, rat, mouse, monkey, and humans); evidence from in vitro studies suggests that the lipid-peroxidizing effects of this enzyme could render the mitochondrion and other intracellular organelles prone to the proteolytic degradation which is a natural step in development of the reticulocyte to the mature red cell. In this study we sought evidence of an active lipoxygenase in vivo. A bleeding anemia was induced in rabbits, and in the course of the subsequent reticulocytosis the red cell membranes were examined for the presence of the characteristic lipoxygenase products of linoleic and arachidonic acids. Erythrocyte membranes from control collections contained only small amounts of hydroxy fatty acids (0.03-0.08% of the polyenoic fatty acids). In contrast, reticulocyte-enriched red cells contained up to 3.3% of the polyenoic acids as hydroxylated derivatives. The main hydroxy fatty acid in reticulocyte membranes was identified as 13-L(S)-hydroxy-9Z,11E-octadecadienoic acid. Small amounts of other hydroxy derivatives including 15-hydroxy-5,8,11,13-(Z,Z,Z,E)eicosatetraenoic acid were also detected. These products appeared about 3 days after development of reticulocytosis. The precise structures of the hydroxylated polyenoic fatty acids and the time course of their appearance strongly suggest that their formation is due to the intracellular action of the cell-specific reticulocyte lipoxygenase. These findings are the first evidence for an activity of this enzyme in vivo, and the results support the hypothesis that enzymic peroxidation of reticulocyte intracellular membranes is a step in preparation of the intracellular organelles for proteolytic degradation.