Endogenous prostacyclin production is best assessed by the measurement of its excreted metabolites, of which a major one is 2,3-dinor-6-ketoprostaglandin F1 alpha (2,3-dinor-6-keto-PGF1 alpha). Gas chromatographic-mass spectrometric (GC-MS) assays have been developed for this compound but are cumbersome and time-consuming. We now report a modified assay for the measurement of 2,3-dinor-6-keto-PGF1 alpha employing GC-MS in which sample preparation time is markedly shortened by replacing a number of extraction steps with reversed-phase column extraction and by modifying derivatization procedures. Precision of the assay is +/- 5% and the accuracy is 98%. The lower limit of detection in urine is approximately 15 pg/mg creatinine. Normal urinary levels of this metabolite were found to be 141 +/- 54 pg/mg creatinine (mean +/- S.D.). Urinary excretion of 2,3-dinor-6-keto-PGF1 alpha is markedly altered in situations associated with abnormalities of prostacyclin generation when quantified using this assay. Thus, this assay provides a sensitive and accurate method to assess endogenous prostacyclin production and to further explore the role of this compound in human health and disease.