A reinvestigation of the structure of mithramycin, the principal product of Streptomyces argillaceus ATCC 12956, is reported. The structure elucidation was carried out with mithramycin decaacetate (4) using 2D NMR methods, including TOCSY, HMBC, and HSQC experiments. The work resulted in structure 3being confirmed for mithramycin.