Fluorescence spectroscopy was used to study the interaction between the minor-groove-binding drug netropsin and the self-complementary oligonucleotide d(CTGAnPTTCAG)2 containing the fluorescent base analogue 2-aminopurine (nP). The binding of netropsin to this oligonucleotide causes strong quenching of the 2-aminopurine fluorescence, observed by steady-state as well as time-resolved spectroscopy. From fluorescence titrations, binding isotherms were recorded and evaluated. The parameters showed one netropsin binding site/oligonucleotide duplex and an association constant of about 10' M -' at 25"C, 3-4 orders of magnitude weaker than for an exclusive adenine/thymine host sequence. From the temperature dependence of the association constant the thermodynamic parameters were obtained as AG = -29 kJ/mol, A H = -12 kJ/mol and A S = + 55 J . mol-' . K-' at 25°C. These parameters resemble those of the interaction of poly [(dG-dC) . (dG-dC)] with netropsin, indicating a mainly entropy-driven reaction. The amino group of 2-aminopurine, like that of guanine, resides in the minor groove of DNA. Therefore the relatively weak binding of netropsin to d(CTGAnPTTCAG), is probably related to partial blockage of the tight fit of netropsin into the preferred minor groove of an exclusive adenine/thymine host sequence.Base-sequence-dependent selectivity and specificity in the interaction of DNA with low-molecular-mass ligands, such as anti-tumour drugs, or with macromolecules such as proteins, is an important biological recognition problem. It is important to understand the detailed molecular background of this recognition and to develop new strategies for experimental studies of specific interactions between DNA and binding ligands. This study concerns the interaction between netropsin and a fluorescent DNA oligomer.Netropsin is an established anti-viral anti-tumour antibiotic (Zimmer and Wiihnert, 1986) which, however, is too toxic for clinical use. Its biological activity is considered to be a consequence of its tight binding to double-helical B-DNA.The interaction of netropsin with DNA has involved a variety of experiments. Foot-printing techniques (Taylor et al., 1984; Dervan, 1986) have established that netropsin binds A + T-rich sites of 5 5 1 base pairs in naturally occurring DNA polymers. A preference for the B conformation of the DNA has been established (Zimmer, 1975). Viscosimetric studies indicate that netropsin binding increases the contour length of A + T-rich DNA helices and makes them more rigid (Reinert et al., 1980). The thermal stability of the DNA is enhanced on netropsin binding and combined spectroscopic and calorimetric techniques have shown that the thermodynamics for netropsin binding to A + T-rich sequences are very favourable, involving an entirely enthalpy-driven interaction Correspondence to A. Grislund,