By combination of several 1H NMR techniques including double quantum filter (DQF)-COSY, relayed-COSY, total correlation spectroscopy (TOCSY), NOESY, and DQF-relayed-NOESY, the sequence of actagardine, a novel lantibiotic produced by Actinoplanes strains ATCC31048 and ATCC31049, was elucidated. It is a tetracyclic 19-residue peptide antibiotic composed of 19 amino acids, with 8 forming 1 lanthionine and 3 β-methyllanthionine units through intramolecular thioether bridges. FAB-mass spectrometry analysis revealed its molecular mass was 16 dalton higher than expected, indicating an additional oxygen atom, leading to the molecular formula C91H124N20O24S4 for actagardine. Modern high-field NMR techniques proved to be a powerful tool for the structure elucidation of actagardine, avoiding amino acid sequencing which was particularly troublesome due to the resistance to enzymatic cleavages and bad reactivity toward the desulfurization treatment required for Edman degradation. Actagardine differs from the previously described lanthionine-containing peptide antibiotics belonging to the lantibiotic class.