We report a convenient and efficient method for the preparation of prostaglandin 2,3-dinor-6-keto-F1 alpha by incubating prostaglandin 6-keto-F1 alpha (6-keto-PGF1 alpha) with dispersed rat hepatocytes. Chromatographic separation revealed a single product from the hepatocyte metabolism of 6-keto-PGF1 alpha whose structure was positively confirmed by mass spectrometry as 2,3 dinor-6-keto-PGF1 alpha. This method allowed for the preparation of high specific activity radioactive 2,3-dinor-6-keto-PGF1 alpha which can be utilized to determine the recovery of urinary dinor-6-keto-PGF1 alpha during extraction and separation of the compound for radioimmunoassay measurements, as well as deuterated 2,3-dinor-6-keto-PGF1 alpha which can be used as an internal standard in the gas chromatography-mass spectrometric assay of this compound.