We determined the cysteine connectivity of protegrin PG-2, a leukocyte-derived antimicrobial peptide, by performing sequential enzyme digestions with chymotrypsin and thermolysin, and monitoring each digest by direct liquid chromatography-electrospray mass spectrometric analysis. This approach resolved the disulphide pairing pattern unambiguously with only picomolar amounts of PG-2. The inferred cysteine connectivity was confirmed by traditional amino acid composition analyses using nanomolar amounts of the protegrin. The results suggest that protegrins will assume a tachyplesin-like, disulphide-stabilized anti-parallel beta-sheet configuration in solution.