Positive and negative ion electrospray mass spectrometry together with Edman sequencing (when appropriate) has been used to sequence the host-defence peptides secreted from skin glands of the tree frog Litoria peronii. The peptide profiles are different in winter and summer. In winter, the frog produces small amounts of the known caerin 1.1 [GLLSVLGSVAKHVLPHVVPVIAEHL-NH2] (a wide-spectrum antibiotic) and caerin 2.1 [GLVSSIGRALGGLLADVVKSKQPA-OH], a narrow-spectrum antibiotic and an inhibitor of neuronal nitric oxide synthase. The major peptides produced throughout the year are the pGlu-containing peroniins 1.1 to 1.5 (e.g. peroniin 1.1 [pEPWLPFG-NH2], a smooth muscle contractor from 10−7 M), and caerulein [pEQDY(SO3H)TGWMDF-NH2], a known and potent smooth muscle contractor from 10−10 M. There are also some precursors to the peroniin 1 peptides, only detected in the skin secretion in summer, which are inactive and appear to be all (or part) of the spacer peroniin 1 peptides, e.g. peroniin 1.1b [SEEEKRQPWLPFG-NH2]. There are three members of the Litoriaperonii Group of tree frogs classified in Australia, namely, L. peronii, L. rothii and L.tyleri. A comparison of the skin peptide profiles of L. peronii with those reported previously for L. rothii suggests that either these two species of tree frog are not as closely related as determined previously on morphological grounds, or that skin peptide divergence in tree frogs of this Group is more extensive than in others that have been studied. Copyright © 2009 John Wiley & Sons, Ltd.