It has been suggested that the amino acid sequence of pancreatic polypeptide (PP) may provide a useful molecular marker with which to study evolutionary relationships between tetrapods but few PP sequences from amphibia are available to test this hypothesis. PPs have been purified from the pancreata of five species belonging to the different orders of amphibians. Their amino acid sequences were established as: APSEPEHPGD10 NASPDELAKY20 YSDLWQYITF30 VGRPRY for the lesser siren, Siren intermedia (Caudata); GPTEPIHPGK10 DATPEELTKY20 YSDLYDYITL30 VGRSRW for the caecilian, Typhlonectes natans (Gymnophiona); and TPSEPQHPGD10 QASPEQLAQY20 YSDLWQYITF30 VTRPRF for the cane toad, Bufo marinus (Anura). The structure of Rana sylvatica PP is the same as that of Rana catesbeiana PP whereas PP from the green frog Rana ridibunda contains one substitution (His6 --> Gln). The data provide further support for the conclusion that the amino acid sequence of PP has been poorly conserved during evolution with only 17 residues invariant among the eight species of amphibia yet studied and only 8 residues (Pro5, Pro8, Gly9, Ala12, Leu24, Tyr27, Arg33, and Arg35) invariant among all tetrapods. A maximum parsimony analysis based upon the amino acid sequence of PP and using the sequence of frog PYY as outgroup to polarize the in-group taxa generates a consensus phylogenetic tree in which the Amniota and Amphibia form two distinct clades. However, such a tree does not permit valid conclusions to be drawn regarding branching order within the Amphibia.