In addition to the highly specific cell-mediated immune system, vertebrates possess an efficient host-defense mechanism against invading microorganisms which involves the synthesis of highly potent antimicrobial peptides with a large spectrum of activity. A 34-residue cationic and amphiphatic peptide, designated dermaseptin I, was recently isolated from the skin of the arboreal frog Phyllomedusa sauvagii and was shown to exhibit microbicidal activity against various pathogenic microorganisms including bacteria, yeast, protozoa and filamentous fungi. In this study, we report the isolation and characterization of four novel antimicrobial peptides from frog skin through the combined use of an anti-dermaseptin enzyme immunoassay and an antifungal bioassay. The 28-34-residue antimicrobial peptides are cationic, containing 3-5 lysine residues that punctuate an alternating hydrophobic and hydrophilic sequence. Based on their primary structure, all four peptides can be fitted to a class L amphipathic alpha helix which places all lysine residues on the polar side of the helix. The four antimicrobial peptides have high sequence similarity with dermaseptin I (53-94% similarity) suggesting that their respective genes are all members of the same family. In addition, pairwise sequence alignment of dermaseptin I and adenoregulin, a 33-residue cationic peptide recently isolated from frog skin and shown to enhance the binding of agonists to the A1 adenosine receptor, reveals 62% similarity (39% amino acid positional identity). Both peptides share a similar but non-identical spectrum of antimicrobial activity, being active against bacteria, yeast and filamentous molds. However, no significant hemolytic activity was found for these peptides which suggests a selectivity for prokaryotic cells. These findings indicate that adenoregulin should be included in the dermaseptin family of peptides. In addition, tryptic digestion of purified pro-dermaseptin I liberated a 15-amino-acid peptide identified as the authentic C-terminus of dermaseptin I. These results are in accordance with the predicted sequences of pro-dermaseptins obtained through molecular cloning, in which the dermaseptin progenitor sequences are located at the extreme C-terminus of the precursors.