The L-to-D-peptide isomerase from the venom of the platypus (Ornithorhyncus anatinus) is the first such enzyme to be reported for a mammal. In the process of delineating its catalytic mechanism and broader roles in the animal, its substrate specificity was explored. We used substrate segments from the N-terminus of the natriuretic peptide (OvCNP) and defensin-like peptides-2 and -4 (DLP-2 and DLP-4) from the venom. Synthetic hexapeptides containing the first three amino acid residues (IMF) of DLP-4 and DLP-2 were linked to the tripeptide srs (lower case letters denote the corresponding D-amino acid), to increase peptide stability and water solubility. The DLP analogues IMFsrs and ImFsrs were found to be effective substrates for the isomerase. Mutants of these hexapeptides were synthesized with the second amino acid replaced, respectively, by the L- and D-forms of Abu, Ala, His, Ile, Leu, Lys, Nle, Phe, Trp, Tyr, and Val. The relative rates of peptide isomerization were measured by using partially purified isomerase extracted from venom glands and separating the reactants with high performance liquid chromatography (HPLC). The isomerase was active with the mutants that contained L- or D-forms of Phe or Nle residues at the second position; but the amino acids that contained shorter, bta-branched- or long side-chains with terminal polar groups, viz., Ala, Abu, Ile, Leu, Val, Lys and Tyr respectively, were not substrates. For each of these non-substrates, newly formed peptides that eluted from HPLC earlier than the substrate were isolated and were deduced to have been formed through the loss of N-terminal Ile in each case. None of the hexapeptides based on LLH, the first three amino acid residues of OvCNPalpha were substrates, but when LLH was attached to full-length OvCNP it became a substrate for the isomerase; thus modulation of the substrate specificity is brought about by other sections of the longer peptide. Based on the action of the isomerase with the various substrates a model of the 'van der Waals outline' of the active site is proposed.