Actinotiocin is a new sulfur-con tainig peptide antibiotic obtained from the cultured broth and the mycelium of Actinomadura pusilla A-118 (S2-118). It is extracted with ethyl acetate and purified by silica gel chromatography. It forms colorless columnar crystals, melts at 247-249°C, and gives [α]20D+164° (c0.77, dioxane). C49H53-55N13O10S5 was suggested for its molecular formula by elemental analysis. On acidic hydrolysis of actinotiocin, a new large molecular amino acid (III, C28H18N6O6S4) having some heteroaromatic rings, serine, proline, glycine and unidentified amino acids were obtained. The partial structure of the amino acid (III) was deduced from the chemical reaction and spectral data. Actinotiocin showed strong antibacterial activity against gram-positive bacteria and no cross resistance with commercial antibiotics. The administration of 1, 000mg/kg of this antibiotic into mice by intraperitoneal and oral routes did not result in any toxic symptoms.