Recent work has shown that oocytes of the starfish synthesize (8R)-hydroxyeicosatetraenoic acid and that this eicosanoid has a potent and highly specific action in induction of oocyte maturation. These striking results prompted us to examine the lipoxygenase activity of eggs of the sea urchin Strongylocentrotus purpuratus. Four hydroxyeicosanoids were formed in homogenates of sea urchin eggs; their structures and stereochemistry were characterized by high pressure liquid chromatography, UV spectroscopy, and gas chromatography-mass spectrometry. The compounds were identified as (11R)-hydroxy-5,8,12,14-ZZEZ-eicosatetraenoic acid and (12R)-hydroxy-5,8,10,14-ZZEZ-eicosatetraenoic acid (from arachidonic acid) and the corresponding (11R)- and (12R)-hydroxy analogs of eicosapentaenoic acid. The formation of these egg products was not blocked by a cyclooxygenase inhibitor, indomethacin (10 microM), and their precise structures are consistent with their formation by a lipoxygenase reaction. Eicosapentaenoic acids with a prochiral tritium label in the 10-D or 10-L position were used to investigate the mechanism of biosynthesis. The formation of (12R)-hydroxyeicosapentaenoic acid proceeded with the stereoselective abstraction of the 10-D hydrogen from the substrate. This reaction was shown to be opposite to the (12S) oxygenation catalyzed by porcine leukocyte 12-lipoxygenase. These results with S. purpuratus eggs constitute the first demonstration of (11R)- or (12R)-lipoxygenase activity in any cell type or tissue.