An assay for the quantitative analysis of six biologically important prostanoids based on combined gas chromatography negative ion chemical ionization mass spectrometry has been developed. Prostanoids were extracted from biological fluids by liquid chromatography on Sep-Pak cartridges and converted to pentafluorobenzyl ester derivatives. Samples were injected on capillary column by the splitless technique and injections were made in a high boiling hydrocarbon solvent (n-dodecane) in order to minimize chromatographic run times. Quantification was carried out using selected ion monitoring of the appropriate [M-pentafluorobenzyl]- anion. The assay has been used for profiling cyclooxygenase metabolites of arachidonic acid in guinea pig lung perfusate after induction of anaphylaxis and platelet rich plasma after collagen-stimulated aggregation.