Four structurally related peptides (pseudins 1-4) with antimicrobial activity were isolated from an extract of the skin of the paradoxical frog Pseudis paradoxa (Pseudidae). Pseudin-2 (GLNALKKVFQGIHEAIKLINNHVQ) was the most abundant peptide (22 nmol/g tissue) and also the most potent (minimum inhibitory concentrations, MIC = 2.5 microM against Escherichia coli, 80 microM against Staphylococcus aureus, and 130 microM against Candida albicans). The concentration of pseudin-2 producing 50% hemolysis of human erythrocytes was >300 microM. Circular dichroism studies showed that the pseudins belong to the class of cationic, amphipathic alpha-helical antimicrobial peptides but their amino acid sequences are not similar to any previously characterized peptides from frog skin. The pseudins do, however, show sequence similarity with a region at the C-terminus of DEFT, a death effector domain-containing protein expressed in mammalian testicular germ cells that is involved in the regulation of apoptosis.