Earlier work showed that C-N bond formation in penicillin biosynthesis from cysteine proceeds with net retention of configuration, but since the process involves conversion of cysteine into the tripeptide L,L,D-ACV followed by cyclisation via isopenicillin N synthetase (IPNS), undetected configurational changes at C-3 of cysteine prior to cyclisation were conceivable. To clarify the stereospecificity of IPNS-catalysed cyclisation, a new method was developed to prepare stereospecifically deuteriated (S)-benzylcysteines, which were used to synthesize deuteriated tripeptide precursors (ACV). These precursors were converted by purified IPNS into isopenicillin N. NMR and mass spectral analyses of the products revealed that the enzymatic conversion of the tripeptide into isopenicillin N occurs with complete retention of the cysteinyl 3-pro-R-hydrogen and complete loss of the cysteinyl 3-pro-S-hydrogen. Additionally, this stereospecificity is unaffected by isotope effects observed in kinetic studies.