A novel peptide with antimicrobial activity was isolated from leukocytes of the European pond turtle Emys orbicularis and purified to homogeneity by preparative gel electrophoresis followed by reversed phase chromatography. It was highly active in vitro against Escherichia coli, Listeria monocytogenes, methicillin-resistant Staphylococcus aureus, and Candida albicans. The isolated peptide was sequenced de novo by tandem mass spectrometry using both collision-induced and electron-transfer dissociation in combination with different chemical derivatization techniques. The 40-residue peptide, called TBD-1 (turtle β-defensin 1), represents the first defensin isolated from reptilian leukocytes. It contains three disulfide bonds and shows high structural similarities to β-defensins isolated from birds and mammals. © 2009 Wiley-VCH Verlag GmbH & Co. KGaA.