We present a detailed conformational study of 15N-labelled actinomycin D in different organic solvents using 1H, 15N and two-dimensional (2D) NMR techniques at 30.4 MHz and 50.6 MHz. The assignment of the threonine and valine 15N resonances to the individual residues on the alpha- or beta-lactone rings was achieved via heteronuclear shift-correlated 2D NMR experiments. The solvent perturbation studies allow an estimation of the solvent accessibility of the nitrogens and carbonyl groups. Evidence is presented that the pentapeptide rings of actinomycin D have different conformations in polar and in apolar solvents. The chromophoric N10 is efficiently solvent-protected, the solvent-dependence of its 15N resonance resulting from solvent interactions at other positions of the molecule and from solvent-dependent changes in the twisting of the chromophoric system. The chromophoric 2-amino nitrogen is shown to exhibit a strong sp2 character due to the formation of a conjugated system with the carbonyl group at C1. Such a conjugation requires a non-planar chromophoric ring system. Additionally, a hydrogen bond connecting the 2-amino and the 1-carbonyl group was detected. In some solvents, two resonances appear for the 2-amino nitrogen implying the presence of the 2-amino group in two different conformations. The possible implications of the non-planarity of the chromophore for the intercalation process and for the biological activity of the drug are discussed.