The complex between distamycin A and the parallel DNA quadruplex [d(TGGGGT)](4) has been studied by H-1 NMR spectroscopy and isothermal titration calorimetry (ITC). To unambiguously assert that distamycin A interacts with the grooves of the quadruplex [d(TGGGGT)](4), we have analyzed the NMR titration profile of a modified quadruplex, namely [d(TGG(Me)GGT)](4), and we have applied the recently developed differential frequency-saturation transfer difference (DF-STD) method, for assessing the ligand - DNA binding mode. The three-dimensional structure of the 4:1 distamycin A/[d(TGGGGT)](4) complex has been determined by an in-depth NMR study followed by dynamics and mechanics calculations. All results unequivocally indicate that distamycin molecules interact with [d(TGGGGT)](4) in a 4:1 binding mode, with two antiparallel distamycin dinners that bind simultaneously two opposite grooves of the quadruplex. The affinity between distamycin A and [d(TGGGGT)](4) enhances (similar to 10-fold) when the ratio of distamycin A to the quadruplex is increased. In this paper we report the first three-dimensional structure of a groove-binder molecule complexed to a DNA quadruplex structure.