Technical Abstract: A new oxyvinylglycine has been identified as a naturally occurring herbicide that irreversibly arrests germination of the seeds of grassy weeds; such as annual bluegrass (Poa annua), without significantly affecting the growth of established grass seedlings and mature plants, or germination of the seeds of broadleaf plant species (dicots). Previously, Pseudomonas fluorescens WH6 and over twenty other rhizosphere bacteria were isolated and selected for their ability to arrest germination of P. annua seeds. The Germination-Arrest Factor (GAF, 1) responsible for this developmentally specific herbicidal action has now been isolated from the culture filtrate of P. fluorescens WH6. Purification of this highly polar, low molecular weight natural product allowed its structure to be assigned as 4-formylaminooxy-Lvinylglycine based on NMR spectroscopic and mass spectrometric data, in combination with D/L-amino acid oxidase reactions to establish the absolute configuration. Assay results for P. annua inhibition by related compounds known to regulate plant growth are presented, and a cellular target for 1 is proposed. Furthermore, using bioassays, TLC, and capillary NMR spectroscopy, it has been shown that GAF (1) is secreted by all other herbicidally-active rhizosphere bacteria in our collection.