In a previous paper we reported that arachidonic acid (20:4( n − 6)) strongly enhances the endothelial cell synthesis of prostaglandin I 3 (PGI 3) from eicosapentaenoic acid (20:5( n − 3)), in stimulating the cyclooxygenase rather than the prostacyclin synthase (Bordet et al. (1986) Biochem. Biophys. Res. Commun. 135, 403–410). In the present study, endothelial cell monolayers were co-incubated with exogenous 20:5( n − 3) or docosatetraenoic acid (22:4( n − 6)), and n − 6 lipoxygenase products of 20:4( n − 6) or linoleic acid (18:2( n − 6)), namely 15-HPETE and 13-HPOD, respectively. Prostaglandins or dihomoprostaglandins were then measured by gas chromatography-mass spectrometry. Both hydroperoxides, up to 20 μM, stimulated the cyclooxygenation of 20:5( n − 3) and 22:4( n − 6), in particular the formation of PGI 3 and dihomo-PGI 3, respectively. Higher concentrations inhibited prostacyclin synthase. In contrast, the reduced products of hydroperoxides, 15-HETE and 13-HOD, failed to stimulate these cyclooxygenations, 13-HPOD appeared more potent than 15-HPETE and the cyclooxygenation of 22:4( n − 6) seemed to require higher amounts of hydroperoxides to be efficiently metabolized than 20:5( n − 3). These data suggest that prostacyclin potential of endothelium might be enhanced by raising the peroxide tone.