Molecular structure and conformational preferences of yatakemycin, a novel and exceptionally potent antitumor agent, have been investigated using the density functional theory (DFT) formalism. From the relative stability of various possible conformations, it is found that two conformers are nearly isoenergetic and markedly more stable than the others in the gas phase. To test the effect of polar mediums, the relative energies have been recalculated using the self-consistent reaction field method. Thus, the most stable conformer of the isolated molecule in the gas phase is expected to be still more preferred in solution. The molecular structure of yatakemycin has also been studied by means of its spectroscopic properties. The DFT results satisfactorily reproduce the experimental data and corroborate the reliability of the structural characterization advanced for yatakemycin. The lowest-energy electronic transitions have been interpreted with time-dependent DFT calculations. Notably, the strong IR band observed at 2852 cm -1 is unambiguously assigned to the O-H stretching of the (C 7)O-H⋯O(C 12) fragment, linked by a strong intramolecular H-bond, and may be viewed as a distinctive fingerprint of yatakemycin. Furthermore, the calculated set of NMR chemical shifts of carbonyl carbon atoms and indole protons, the most sensitive to stereoelectronic factors, is consistent with experiment. The effects of both protonation and oxidation on the geometry of the most stable conformer have also been studied. With reference to yatakemycin's DNA alkylation properties, the structure of the yatakemycin-adenine adduct has been theoretically modeled and found to be consistent with experimental spectroscopic evidence.