Negamycin was isolated from strains related to Streptomyces purpeofuscus. In the early growth phase of the negamycin-producing strain Streptomyces No. M890-C2, a new antibiotic leucylnegamycin was identified. It was isolated by resin chromatography and could be converted to negamycin by leucine aminopeptidase or washed cells of the strain, suggesting it is an intermediate in negamycin biosynthesis. This study focused on its isolation, characterization, and structure determination. Leucylnegamycin is a colorless powder with m.p. 127-133°C (decomp.), [α]²⁶_D +4.8° (c 2, H₂O), and molecular formula C₁₅H₃₁N₅O₅·1/2H₂O. Its structure was determined as [2-{3(R)-amino-5(R)hydroxy-6-L-leucylaminohexanoyl}-1-methylhydrazino]acetic acid via analyses of hydrolysis products, NMR, IR, high-resolution mass spectrometry, and enzymatic confirmation of the L-configuration of leucine. It exhibits weaker antimicrobial activity than negamycin. Experiments with L-leucine-(U)-¹⁴C confirmed it is a direct metabolic intermediate in negamycin biosynthesis, similar to leucylblasticidin S.