In the course of our screening program for novel antimetabolites competitive with L-glutamine from soil actinomycetes, a new herbicidal antibiotic was isolated from the culture filtrate of Kitasatosporia phosa/acinea sp. nov. KA-338. This communication describes the structural elucidation of this new phosphorus-containing tripeptide herbicide (phosalacine). Through physicochemical analyses including melting point (225°C dec), specific rotation ([α]D²⁰ 38.8°), FD-MS (m/z 366 (M-1)⁺), high-resolution EI-MS (confirming molecular formula C₁₇H₂₈N₃O₇P), IR (indicating carboxylic acid and amide carbonyl groups), ¹³C NMR (suggesting a methylphosphorous-methylene skeleton), acid hydrolysis amino acid analysis (revealing L-alanine, L-leucine, and L-phosphinothricin), TLC comparison, and EI-MS (determining the amino acid sequence as phosphinothricylalanylleucine), the structure of phosalacine was finally determined.