11-Dehydro-thromboxane B 2 has been identified as a major metabolite of infused as well as endogenous thromboxane B 2 in mammalian plasma and urine. This metabolite is derived from thromboxane B 2 by enzymatic oxidation at C-11 catalyzed by 11-hydroxytrhomboxane B 2 dehydrogenase. A radioimmunoassay for 11-dehydro-thromboxane B 2 has been developed and used for enzyme assay, purification and characterization. Antibodies were generated against 11-dehydrothromboxane B 2 conjugated to bovine thyroglobulin. Labeled marker was prepared by radioiodinating 11-dehydrothromboxane B 2-tyrosine methyl ester conjugate. A sensitive radioimmunoassay capable of detecting 10 pg of 11-dehydro-thromboxane B 2 per assay tube was developed. The antibodies showed minimal crossreaction with thromboxane B 2 (0.03%), prostaglandin D 2 (2.76%) and other eicosanoids (<0.03%). The enzyme activity was determined by assaying NAD +-dependent formation of immunoreactive 11-dehydro-thromboxane B 2 from thromboxane B 2. The enzyme was found to be enriched in liver although significant activity was also detected in gastrointestinal tract and kidney in pig. The enzyme was purified from porcine liver cytosol to apparent homogeneity using conventional and affinity chromatography. The purfied enzyme exhibited coenzyme specificity for NAD + and used thromboxane B 2 as a substrate. The enzyme also catalyzes NADH-dependent reduction of 11-dehydro-thromboxane B 2 to thromboxane B 2 indicating the reversibility of the enzyme catalyzed reaction. The apparent K m values for thromboxane B 2, 11-dehydrothromboxane B 2 and NAD + are 8.1, 8.0 and 23 μM, respectively. Subunit M r was shown to be 55 000, whereas the native enzyme M r was found to be 110 000 indicating that the enzyme is a dimer. The enzyme is sensitive to sulfhydryl inhibitions suggesting cysteine residues are essential to enzyme activity. The availability of a homogeneous enzyme preparation should allow further studies on the substrate specificity and the structure and function of the enzyme.