The biosynthesis of the phenazine antibiotics, the esmeraldins and the related saphenamycins, was studied by feeding radioactive and stable isotope-labeled precursors to Streptomyces antibioticus, strain TU 2706. After purification, the labeled antibiotics were degraded to the diastereomeric esmeraldic acid dimethyl esters 3 and 4 and racemic saphenic acid methyl ester (51, respectively. The 'H- and W-NMR spectra of these were assigned by single and multiple bond correlation experiments. Although 1%- and 13C-labeled shikimic acid was not incorporated, W-NMR analysis of the samples biosynthesized from [U-W3]-, [ lI3J3C2]-, and [2-'3Clglycerol provided evidence that these phenazine antibiotics are derived from the shikimic acid pathway and display the same head-to-tail coupling pattern of monomer units seen in other phenazine antibiotics. The extra methyl group attached to the carbon corresponding to the shikimate carboxyl group originates from C-2 of acetate. Radiolabeled saphenic acid was incorporated into the diphenazine ring system of the esmeraldins. Mechanistic aspects of the formation of the phenazine and diphenazine ring systems are discussed.