Stable isotope labeling experiments have shown that thr biosynthesis of the monomeric phenazines, the saphenyl esters, and their dimerization products, the esmeraldins, in Strepromyces antibioticus Tu 2706 proceeds from phenazine-1,6-dicarboxylic acid by chain extension with C-2 of acetate to 6-acetylphenazine-1-carboxylic acid, which is reduced to saphenic acid. The latter is incorporated into both halves of the esmeraldins, albeit differentially. By feeding of chiral acerate, degradation of the resulting saphenyl esters and esmeraldins, and configurational analysis of the acetic acid formed, the chain extension process was found to proceed with overall inversion of configuration at the methyl group. This suggests that the decarboxylation of a hypothetical intermediate beta-keto acid proceeds in an inversion mode. This result is discussed with reference to analogous C-methylations of polyketide backbones by addition of C-2 of acetate,