The biosynthesis of syringomycin (SR) and syringopeptin 22 (SP22), bioactive lipodepsipeptides of the phytopathogenic bacterium Pseudomonas syringae pv. syringae, was studied by feeding (14)C-labeled precursors to chloramphenicol-containing bacterial suspensions. The preferential sites of incorporation were determined by comparing the specific activities of the intact radiolabeled metabolites and their single structural elements, obtained by hydrolytic degradation followed by derivatization and isolation by high performance liquid chromatography. The results show that, upon feeding L-[(14)C(U)]-Thr, 35.0 and 31.0% of the SR radioactivity is retained in 2,3-dehydro-2-aminobutyric acid (Dhb) and 4-chlorothreonine (Thr(4-Cl)), respectively. L-[(14)C(U)]-Asp labels the same sites, though less efficiently, and is also incorporated in 2,4-diaminobutyric acid (Dab) and 3-hydroxyaspartic acid (Asp(3-OH)). Dhb is also labeled by Thr and Asp in SP22. These are the first data on the biosynthetic origin of the modified residues in P. syringae lipopeptides.