Protectin D1, neuroprotectin D1 when generated by neural cells, is a member of a new family of bioactive products generated from docosahexaenoic acid. The complete stereochemistry of protectin D1 (10, 17S-docosatriene), namely, chirality of the carbon-10 alcohol and geometry of the conjugated triene, required for bioactivity remained to be assigned. To this end, protectin D1/neuroprotectin D1 (PD1) generated by human neutrophils during murine peritonitis and by neural tissues was separated from natural isomers and subjected to liquid chromatography-tandem mass spectrometry and gas chromatography-mass spectrometry. Comparisons with six 10, 17-dihydroxydocosatrienes prepared by total organic and biogenic synthesis showed that PD1 from human cells carrying potent bioactivity is 10R, 17S-dihydroxy-docosa-4Z, 7Z, 11E, 13E, 15Z, 19Z-hexaenoic acid. Additional isomers identified included trace amounts of Δ15-trans-PD1 (isomer III), 10S, 17S-dihydroxy-docosa-4Z, 7Z, 11E, 13Z, 15E, 19Z-hexaenoic acid (isomer IV), and a double dioxygenation product 10S, 17S-dihydroxy-docosa-4Z, 7Z, 11E, 13Z, 15E, 19Z-hexaenoic acid (isomer I), present in exudates. 18 O 2 labeling showed that 10S, 17S-diHDHA (isomer I) carried 18 O in the carbon-10 position alcohol, indicating sequential lipoxygenation, whereas PD1 formation proceeded via an epoxide. PD1 at 10 nM attenuated (∼ 50%) human neutrophil transmigration, whereas Δ15-trans-PD1 was essentially inactive. PD1 was a potent regulator of polymorphonuclear leukocyte (PMN) infiltration (∼ 40% at 1 ng/mouse) in peritonitis. The rank order at 1-to 10-ng dose was PD1≈ PD1 methyl ester≫ Δ15-trans-PD1> 10S, 17S-diHDHA (isomer I). 10S, 17S-dihydroxy-docosa-4Z, 7Z, 11E, 13E, 15Z, 19Z-hexaenoic acid (isomer VI) proved≥ PD1 in blocking PMN infiltration, but was not a major product of leukocytes. PD1 also reduced PMN infiltration after initiation (2 h) of inflammation and was additive with resolvin E1. These results indicate that PD1 is a potent stereoselective anti-inflammatory molecule.