Two neuropeptides have been isolated and identified from the secretions of the skin glands of the Stony Creek Frog Litoria lesueuri. The first of these, the known neuropeptide caerulein 1.1, is a common constituent of anuran skin secretions, and has the sequence pEQY(SO3)TGWMDF-NH2. This neuropeptide is smooth muscle active, an analgaesic more potent than morphine and is also thought to be a hormone. The second neuropeptide, a new peptide, has been named lesueurin and has the primary structure GLLDILKKVGKVA-NH2. Lesueurin shows no significant antibiotic or anticancer activity, but inhibits the formation of the ubiquitous chemical messenger nitric oxide from neuronal nitric oxide synthase (nNOS) at IC(50) (16.2 microm), and is the first amphibian peptide reported to show inhibition of nNOS. As a consequence of this activity, we have tested other peptides previously isolated from Australian amphibians for nNOS inhibition. There are three groups of peptides that inhibit nNOS (IC(50) at microm concentrations): these are (a) the citropin/aurein type peptides (of which lesueurin is a member), e.g. citropin 1.1 (GLFDVIKKVASVIGGL-NH(2)) (8.2 microm); (b) the frenatin type peptides, e.g. frenatin 3 (GLMSVLGHAVGNVLG GLFKPK-OH) (6.8 microm); and (c) the caerin 1 peptides, e.g. caerin 1.8 (GLFGVLGSIAKHLLPHVVPVIAEKL-NH(2)) (1.7 microm). From Lineweaver-Burk plots, the mechanism of inhibition is revealed as noncompetitive with respect to the nNOS substrate arginine. When the nNOS inhibition tests with the three peptides outlined above were carried out in the presence of increasing concentrations of Ca(2+) calmodulin, the inhibition dropped by approximately 50% in each case. In addition, these peptides also inhibit the activity of calcineurin, another enzyme that requires the presence of the regulatory protein Ca(2+) calmodulin. It is proposed that the amphibian peptides inhibit nNOS by interacting with Ca(2+)calmodulin, and as a consequence, blocks the attachment of this protein to the calmodulin domain of nNOS.