Peptidomics　is　a　powerful　set　of　tools　for　the　identification,　structural　elucidation　and　discovery　of　novel　regulatory　peptides　and　for　monitoring　the　degradation　pathways　of　structurally　and　catalytically　important　proteins.　Amphibian　skin　secretions,　arising　from　specialized　granular　glands,　often　contain　complex　peptidomes　containing　many　components　of　entirely　novel　structure　and　unique　site-substituted　analogues　of　known　peptide　families.　Following　the　discovery　that　the　granular　gland　transcriptome　is　present　in　such　secretions　in　a　PCR-amenable　form,　we　designed　a　strategy　for　peptide　structural　characterization　involving　the　integration　of　＇shotgun＇　cloning　of　cDNAs　encoding　peptide　precursors,　deduction　of　putative　bioactive　peptide　structures,　and　confirmation　of　these　structures　using　tandem　MS/MS　sequencing.　Here,　we　illustrate　this　strategy　by　means　of　elucidation　of　the　primary　structures　of　nigrocin-2　homologues　from　the　defensive　skin　secretions　of　four　species　of　Chinese　Odorrana　frogs,　O.　schmackeri,　O.　livida,　O.　hejiangensis　and　O.　versabilis.　Synthetic　replicates　of　the　peptides　were　found　to　possess　antimicrobial　activity.　Nigrocin-2　peptides　occur　widely　in　the　skin　secretions　of　Asian　ranid　frogs　and　in　those　of　the　Odorrana　group,　and　are　particularly　well-represented　and　of　diverse　structure　in　some　species.　Integration　of　the　molecular　analytical　technologies　described　provides　a　means　for　rapid　structural　characterization　of　novel　peptides　from　complex　natural　libraries　in　the　absence　of　systematic　online　database　information.　©　2010　FEBS.