Our search for an inhibitor of thermolysin (a metalloendopeptidase from Bacillus thermoproteolyticus) yielded phosphoramidon, which was isolated from culture filtrates of Streptomyces tanashiensis strain MD706-Y4. The production and isolation were studied in detail: the inhibitor was produced in media containing glycerol, meat extract, polypeptone, yeast extract, NaCl, MgSO₄·7H₂O, K₂HPO₄, and CaCO₃ (pH 7.4), with maximum production attained in 2-3 days by rotary culture. Purification involved adsorption on activated carbon, elution with methanol (pH 8.0), evaporation, DEAE-Sephadex A-25 column chromatography (linear NaCl gradient 0-1.0M in 1M acetic acid), ethanol extraction, and Sephadex LH-20 chromatography. The sodium salt of the inhibitor was obtained, and its properties were determined: m.p. 173-178°C (dec.), [α]D²⁰ -33.6° (c. 1.0, H₂O), UV maxima at 221, 275 (sh.), 282, 289.5 nm, IR spectrum, elemental analysis (calcd. for C₂₃H₃₂N₈O₁₀P₁Na₂: C 47.02, H 5.49, N 7.15, O 27.23, P 5.27, Na 7.82; found: C 46.98, H 5.46, N 7.11, O 27.30, P 5.44), positive Ehrlich, ammonium molybdate-perchloric acid, and Rydon-Smith reactions (negative ninhydrin), specific thin-layer chromatography Rf values, and anode migration in electrophoresis. Identity with phosphoramidon was confirmed by comparison with an authentic sample. Inhibition tests showed phosphoramidon is a specific inhibitor of thermolysin (ID₅₀ 0.4μg/ml) with no inhibition of trypsin, papain, α-chymotrypsin, or pepsin (ID₅₀ >250μg/ml). It has no antibacterial/antifungal activity and low toxicity (1.0g/kg intravenous injection in mice caused no death). The specific action suggests interaction between the phosphoric acid moiety of phosphoramidon (N-(α-L-rhamnopyranosyloxyhydroxyphosphinyl)-L-leucyl-L-tryptophan) and Zn²⁺ in the thermolysin active site.