The metabolism of prostaglandin E2 (PGE2) in isolated rat hepatocytes led to the formation of four major as well as several minor products which were structurally characterized using electrospray tandem mass spectrometry. The major metabolites identified included dinor-PGE1, dinor-PGE2, and tetranor-PGE1 and the taurine conjugates of dinor-PGE1 and dinor-PGE2. Several minor metabolites including the taurine conjugates of PGE2 and tetranor PGE1 along with a glucuronide conjugate of PGE2 were also identified. These taurine conjugates had not been previously identified in studies of PGE2 metabolism, yet comprised nearly 50% of the mixture of metabolites after 40-min incubations. Experiments carried out with deuterium-labeled PGE2 ([3,3,4,4-D4]PGE2) resulted in the complete loss of all deuterium atoms in dinor-PGE1, dinor-PGE2, and tetranor metabolites during incubation with hepatocytes. Metabolism via classic beta-oxidation pathways would predict one deuterium atom retained by dinor-PGE1 and two deuterium atoms retained by dinor-PGE2. When PGE2 was incubated with isolated rat hepatocytes in buffer containing 30% D2O, substantial incorporation (30%) of one deuterium atom could be observed in the dinor metabolites along with 10% incorporation into the tetranor and residual PGE2. Deuterium-labeled PGE1 ([3,3,4,4-D4]PGE1) was metabolized to D2-dinor-PGE1, tetranor-PGE1, and the taurine conjugate of D2-dinor-PGE1 by isolated rat hepatocytes. The loss of deuterium during metabolism of the deuterated substrates of PGE2, but not PGE1, as well as the incorporation of deuterium atoms from the aqueous solvent into PGE2 metabolites suggested that the delta 5 double bond and sequential isomerization reactions lead to eventual exchange of the protons from carbon atom 4 of PGE2 with water.