In 1962, one of the authors (Hata) developed a novel screening method using Chlorella to identify plant growth regulators in metabolites of soil microbes. This study reports the isolation and structural elucidation of Fragin, a new plant growth inhibitor produced by a soil bacterium identified as Pseudomonas fragi. The bacterium was cultured in a medium containing 0.02% K2HPO4, 0.05% peptone, 0.02% yeast extract, and 1% glucose at 26°C for 10 days. The culture broth was adjusted to pH 5 with dilute sulfuric acid and extracted with benzene. After drying over anhydrous sodium sulfate, the extract was concentrated, washed with 5% sodium bicarbonate, and extracted with 5% sodium carbonate. The aqueous layer was acidified to pH 5 and re-extracted with benzene. The extract was dried, evaporated, and crystallized from n-hexane to obtain crude Fragin. Further purification via cupric salt precipitation, hydrogen sulfide treatment, and recrystallization yielded pure Fragin as colorless plates with a melting point of 80°C and [α]D -122° (c=1.9 in ethanol). Elemental analysis and molecular weight determinations assigned the molecular formula C13H27O3N2Na (calcd: C 57.11%, H 9.96%, N 15.37%; found: C 56.94%, H 9.82%, N 15.28%). Spectroscopic data (UV: 230 nm in ethanol, 248 and 300 nm in 0.1 N NaOH; IR: 3440, 1680, 1470, 1060 cm⁻¹ in CCl₄) and color reactions (red with ferric chloride, red with phenol and concentrated sulfuric acid) indicated the presence of an N-nitrosohydroxylamino group. Hydrolysis of Fragin produced caprylic acid, suggesting an amide group. NMR analysis of the ketone derivative (IV) revealed an -NH-CH₂-CO-CH(CH₃)₂ moiety. The structure was confirmed by the synthesis of racemic Fragin (to be reported separately). Biologically, Fragin inhibited Chlorella growth at 20 ppm, lettuce seedling root growth at 10 ppm and hypocotyl expansion at 25 ppm, and Aspergillus niger growth at 20 ppm.