The reaction of 5 S,15 S-dihydroperoxyeicosatetraenoic acid (5,15-diHpETE) with human 5-lipoxygenase (LOX), human platelet 12-LOX, and human reticulocyte 15-LOX-1 was investigated to determine the reactivity and relative rates of producing lipoxins (LXs). 5-LOX does not react with 5,15-diHpETE, although it can produce LXA when 15-HpETE is the substrate. In contrast, both 12-LOX and 15-LOX-1 react with 5,15-diHpETE, forming specifically LXB. For 12-LOX and 5,15-diHpETE, the kinetic parameters are k = 0.17 s and k/ K = 0.011 μM s [106- and 1600-fold lower than those for 12-LOX oxygenation of arachidonic acid (AA), respectively]. On the other hand, for 15-LOX-1 the equivalent parameters are k = 4.6 s and k/ K = 0.21 μM s (3-fold higher and similar to those for 12-HpETE formation by 15-LOX-1 from AA, respectively). This contrasts with the complete lack of reaction of 15-LOX-2 with 5,15-diHpETE [Green, A. R., et al. (2016) Biochemistry 55, 2832-2840]. Our data indicate that 12-LOX is markedly inferior to 15-LOX-1 in catalyzing the production of LXB from 5,15-diHpETE. Platelet aggregation was inhibited by the addition of 5,15-diHpETE, with an IC of 1.3 μM; however, LXB did not significantly inhibit collagen-mediated platelet activation up to 10 μM. In summary, LXB is the primary product of 12-LOX and 15-LOX-1 catalysis, if 5,15-diHpETE is the substrate, with 15-LOX-1 being 20-fold more efficient than 12-LOX. LXA is the primary product with 5-LOX but only if 15-HpETE is the substrate. Approximately equal proportions of LXA and LXB are produced by 12-LOX but only if LTA is the substrate, as described previously [Sheppard, K. A., et al. (1992) Biochim. Biophys. Acta 1133, 223-234].