The enzyme isopenicillin N synthetase is able to convert directly the dipeptides phenoxyacetylcysteinylvaline and phenylacetylcysteinylvaline into penicillin V and G respectively, though very slowly compared with substrates of the α-aminoadipoyl or adipoylcysteinylvaline type. With pure IPNS, it was found that peptides containing aromatic and unsaturated moieties instead of the α-aminoadipoyl side chain can be cyclised to corresponding penicillins at very low rates. Incubation of phenoxyacetyl-L-cysteinyl-D-valine with IPNS produced penicillin V methyl ester (0.04% conversion) and phenylacetyl-L-cysteinyl-D-valine produced penicillin G methyl ester (0.12% conversion), identified by gas chromatography-mass spectrometry. Kinetic parameters (Michaelis constants and maximum velocity) revealed a major effect on Vmax, indicating that the catalytic event (rather than binding) is sensitive to the absence of the 6-carboxy group, which substantially depresses conversion rates.