Streptonigrin (1), a potent anticancer antibiotic isolated from Streptomyces flocculus in 1959, was previously shown via biosynthetic experiments to derive its A-ring from the shikimic acid pathway, with 4-aminoanthranilic acid (2) specifically incorporated into this portion—providing the first indication that 2 may be a natural product. We now report that 2 is indeed produced by S. flocculus. Two 50-ml fermentation broths of S. flocculus were inoculated and fed with d-[1-¹⁴C]erythrose. Authentic 2 was added as a carrier to trap de novo synthesized radioactive 2 from one broth terminated 2 hours after erythrose addition (the other served as a control). Following workup, isolation, purification, and acetylation to form bis-acetamide 4, recrystallized samples exhibited specific molar radioactivities indicating a 0.22% incorporation of erythrose into 2. Since establishing a previously unknown compound as a true biosynthetic intermediate requires demonstrating its production by the relevant organism, this study confirms 4-aminoanthranilic acid as a natural product of S. flocculus.