A new method of analysis has allowed the exonucleolytic cleavage by human T-exonuclease to be determined. Hydrolysis by human plasma T-exonuclease proceeds with retention of configuration at phosphorus. The new method determines the sense of chirality at phosphorus in isotopomeric adenosine 5'-O-[O-18]phosphorothioates. This is based on stereospecific two-step conversion of the mono-thionucleotide into the corresponding deoxyadenosine 5'-O-alpha-[O-18]thiotriphosphate, followed by the use of terminal deoxyribonucleotidyl transferase and MALDI TOF mass spectrometry of the resulting elongated primer. Retention of configuration in the reaction of plasma T-exonuclease implies a two-step mechanism with two displacements on phosphorus. Inversion at each step leads to overall retention.