In 1983 Hopwood et al. isolated a calcium-dependent antibiotic (CDA) from Streptomyces coelicolor A3(2) that inhibits the growth of gram-positive bacteria in the presence of Ca²⁺ ions and forms channels selectively transporting singly charged cations. Different culture media yielded various CDA components: yeast extract as a nitrogen source provided CDA1 (Mᵣ=1562) and CDA2 (Mᵣ=1576), while soy peptone and meat extract gave CDA3a (Mᵣ=1480), CDA3b (Mᵣ=1482), CDA4a (Mᵣ=1494), and CDA4b (Mᵣ=1496). Hydrolysis and chromatographic-mass spectrometric analysis revealed amino acid composition including L-ASX, Gly, L-Ser, L-Thr, a 1:1 ratio of L- and D-Trp, and unusual residues such as D-4-hydroxyphenylglycine, D-3-hydroxyaspartic acid (2R:3S), and 3-methylglutamic acid. The N-terminus of CDA2 was identified as 2,3-epoxyhexanoyl via HMBC NMR. Sequence determination using NOESY NMR and Edman degradation (after BNPS-skatole cleavage) showed a lactone ring between the C-terminal tryptophan and threonine at position 2. CDA2 contains D-3-phosphohydroxyasparagine, which is reported here for the first time in a natural compound. CDA1 differs from CDA2 by replacing 3-methylglutamic acid with glutamic acid. CDA3a/b and CDA4a/b are non-phosphorylated, substituting D-3-phosphohydroxyasparagine with D-3-hydroxyasparagine. Conformational studies of CDA in the presence of Ca²⁺ are ongoing.