Streptonigrin (1), a potent anticancer antibiotic isolated from Streptomyces flocculus in 1959, has been shown via biosynthetic experiments to have its A-ring derived from the shikimic acid pathway. Previous work reported the specific incorporation of 4-aminoanthranilic acid (2) into this A-ring, providing the first indication that 2 may be a natural product. Here, we demonstrate that 2 is indeed produced by S. flocculus. Two 50-ml fermentation broths were inoculated, and d-[1-¹⁴C]erythrose was added to each flask. Authentic 2 (synthesized from 4-nitroanthranilic acid) was added as a carrier to trap de novo produced radioactive 2 prior to workup. Workup included pH adjustment, sonication, centrifugation, filtration, lyophilization, trituration with methanol, concentration, acetylation, and recrystallization. Specific molar radioactivities of recrystallized 2 (2.59–2.78 × 10⁴ dpm/mmol) confirmed incorporation of the radioactive label, with a 0.22% incorporation rate from the d-[1-¹⁴C]erythrose (1.18 × 10⁷ dpm/mmol). This result establishes that 4-aminoanthranilic acid (2), a previously unknown natural product, is produced by S. flocculus, fulfilling the requirement for it to be a true biosynthetic intermediate.