We have been screening microbial fermentation for inhibitors of the mitogenic activity of epidermal growth factor. During the screening, a new cyclic lipopeptide antibiotic, enamidonin, which exhibits detransforming activity against a rat cell line transformed with a temperature-sensitive Rous sarcoma virus (srcfs-NRK cells), was isolated from the culture broth of Streptomyces sp. 91-75. This producing strain was isolated from a soil sample collected in Imaichi city, Tochigi prefecture, and deposited at the National Institute of Bioscience and Human-Technology under the accession number FERMP-14190. Enamidonin was purified through a series of steps including extraction with 70% aqueous acetone and ethyl acetate, silica gel column chromatography, Sephadex LH-20 chromatography, and preparative HPLC, yielding a white powder. The antibiotic decomposes at 180–220°C, has an optical rotation of [α]D²⁰ +20° (c 0.03, methanol), and is soluble in dimethyl sulfoxide (DMSO), acetone, and methanol, sparingly soluble in chloroform, and insoluble in water or hexane. It gave positive reactions to ninhydrin and anisaldehyde-H₂SO₄ tests. Amino acid analysis revealed the presence of glycine, L-phenylalanine, and 2,3-diaminopropionic acid. Mass spectrometry data (SI-MS and HRFAB-MS) established the molecular formula as C₃₇H₅₁N₇O₇. Detailed 2D NMR experiments identified structural moieties including N,N-isopropylidene, (E)-3-aminopropenoic acid, and (2E;4E;9E)-13-hydroxytetradeca-2,4,9-trienoic acid. Enamidonin inhibited EGF-dependent [³H]thymidine uptake into Balb/MK cells with an IC₅₀ of 10 μg/ml and reversed the transformed morphology of srcfs-NRK cells to a normal flat morphology with an ED₅₀ of 10 μg/ml. However, it showed no inhibitory activity against tested fungi and bacteria at a concentration of 4 μg/disk using the paper disk-agar method. Detailed studies on the biological activity and mechanism of action of enamidonin are ongoing.