Phosmidosine is a new antifungal proline-containing nucleotide antibiotic recently isolated from culture filtrates of Streptomyces durhameusis, which exhibits specific inhibitory activity against spore formation of Botrytis cinerea, a world-wide pathogenic fungus causing gray mold disease in a variety of fruits and vegetables. The structure of phosmidosine (1), the compound on which biological testing was carried out, was determined by mass spectrometry and NMR spectroscopy. Homologs 2, 3, and the isomer 4 were detected and characterized using approaches based principally on tandem mass spectrometry and combined liquid chromatography-mass spectrometry, which permitted assignment of most structural features directly in the crude isolate without prior isolation of individual components. Conversion of 1-4 to the O-isopropylidene derivatives la-4a by a microscale procedure resulted in enhanced fast atom bombardment ionization (FAB) signal-to-background sensitivity. Collision-induced dissociation mass spectra were acquired from molecular ions and ion source-generated fragment ions and used in conjunction with FAB-deuterium exchange methods for the assignment of structural differences between la-4a.