The biosynthesis of 4-hydroxy-3-nitrosobenzamide in Streptomyces murayamaensis mutants MC2 and MC3 has been studied using sodium [1,2-C-13(2)]- and [1-C-13,O-18(2)]acetate, sodium [2,3-C-13(2)]succinate, [1,2-C-13(2)]glutamic acid, [4-C-13]aspartic acid, and sodium [1-C-13]- and [2,3-C-13(2)]pyruvate. C-13 NMR analysis of the labeling patterns from the first two of these suggested a pathway via condensation of a four-carbon unit from the tricarboxylic acid (TCA) cycle with a three-carbon unit, possibly phosphoenol pyruvate. Subsequent specific incorporations of the labeled succinic acid, aspartic acid, and glutamic acid confirmed the TCA cycle involvement and the orientation of the four-carbon intermediate. Specific incorporation of the labeled pyruvic acids confirmed the involvement of a three-carbon unit and defined its orientation. This is the first aminohydroxybenzoic acid derivative shown not to be derived from a shikimic acid-type pathway, and its origin provides a rationale for the biosynthesis of other microbial products such as asukamycin, manumycin, and the michigazones.