We have developed a direct enzyme immunoassay (EIA) for quantifying immunoreactive 11-keto-thromboxane B2 (iKTXB) in unprocessed human urine. Cross-reactivity with other thromboxane metabolites and prostanoids was negligible. Analytical recovery of 11-keto-TXB2 in urine specimens was 97.4% to 99.8%. Total imprecision for two clinical specimens was 8.5% and 12.2%. Intake of acetylsalicylic acid decreased the measured concentration of iKTXB. Cardiopulmonary bypass, a procedure known to activate platelets, increased the mean excretion rate of iKTXB 10-fold. Simultaneous gas chromatography-mass spectrometry analysis of 11-keto-TXB2 and 11-keto-2,3-dinor TXB2 in urine specimens (n = 17) from healthy subjects indicated that urinary iKTXB concentrations measured by EIA represented a sum of the two 11-keto metabolites. We conclude that the direct EIA is sufficiently sensitive, rapid, simple, and specific to allow screening for alterations in thromboxane biosynthesis in patients.