11-Dehydro-2,3-dinorthromboxane B3 (an enzymatic metabolite of TXB3) was identified. Urine from a healthy male adult who had received daily administration of 1.8 g (0.6 g three times) of eicosapentaenoic acid (EPA) ethyl ester was collected. A urine sample was extracted with an affinity column of anti-11-dehydro-TXB2 monoclonal antibody. Conversion of 11-dehydro-2,3-dinor-TXB3 in the urine extract into the methyl ester (ME)-11-n-propylamide (PA)-9, 12, 15-dimethylisopropylsilyl (DMIPS) ether derivative was followed by gas chromatography with selected-ion monitoring (GC/SIM) (resolution: 8000) and GC/mass spectrometry (MS). High-resolution SIM result revealed that a major component corresponded to the [M - C3H7]+ (m/z 668.4198) ion, which may be specific for the structural integrity by analogy with the corresponding 11-dehydro-TXB3 derivative. Conventional GC/MS analysis confirmed that the peak of the interest was the title compound. The mass spectrum of the 11-dehydro-2,3-dinor-TXB3 ME-PA-DMIPS ether derivative was dominated by the ions of [M - CH3]+ (m/z 696) and [M - C3H7]+ (m/z 668, base peak) and ions containing a protected 11-carboxylic acid moiety together with the unidentified additional ions which were considered to derive from urinary endogenous substances. However, the quality was sufficient for use of mass spectral identification of 11-dehydro-2,3-dinor-TXB3, the beta-oxidation product of 11-dehydro-TXB3. Characteristic fragment ions of [M - C5H9-(dimethylisopropylsilanol)n]+ (m/z 524 and 406) yielding information about the position of the incorporated double bond were prominent, the same as the corresponding ions found in the mass spectrum of the 11-dehydro-TXB3 derivative. Consequently, the fragmentation products were closely related to those of the 11-dehydro-TXB3 derivative, except for an obvious shift produced by the lack of C-2/C-3 hydrocarbon units. The results demonstrate the endogenous formation of TXA3 in humans after administration of EPA ethyl ester and excretion of 11-dehydro-2,3-dinor-TXB3 into urine as one of the enzymatic metabolites of TXB3, together with the previously reported 11-dehydro-TXB3.