(R)- and (S)-2-amino[2-D1]ethylphosphonic acids ([2-D1]AEP) were synthesised to investigate the stereochemistry of the reaction catalysed by 2-aminoethlphosphonate aminotransferase from Pseudomonas aeruginosa. This enzyme catalyses the transfer of the amino group of AEP to pyruvate to produce 2-phosphonoacetaldehyde and alanine. The enzymic reaction proceeding through the abstraction of a proton from the Schiff-base complex formed between the enzyme-bound pyridoxal 5'-phosphate, and the substrate, was carried out in an aqueous buffer at pH 8.5; it was followed by high-field 1H-NMR measurements (500 MHz, H2O) on an AMX 500 Bruker spectrometer. The spectra, recorded with chiral (R)- or (S)-[2-D1]AEP, both showed the methylenic signal (3.0 ppm), whereas (S)-[2-D1]AEP gave the additional aldehydic signal (CHO, 9.6 ppm). These data clearly show that AEP-aminotransferase catalyses the abstraction of the pro-S hydrogen atom at the prochiral C2 carbon of AEP. Furthermore, careful timing of NMR measurements over a 2-hour period allows us to show the occurrence of an isotopic effect.