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500 MHz NMR spectroscopy has been used to investigate the complexation of the anthracycline antibiotic daunomycin (DAU) with self-complementary deoxyhexanucleotides, 5`-d(CGTACG) and 5`-d(CGCGCG), of different base sequence in aqueous salt solution. 2D homonuclear 1H NMR spectroscopy (TOCSY and NOESY) and heteronuclear 1H 31P NMR spectroscopy (HMBC) have been used for complete assignment of the non-exchangeable protons and the phosphorus resonance signals, respectively, and for a qualitative determination of the preferred binding sites of the drug. Analysis shows that DAU intercalates preferentially into the terminal d(CpG) site of each of the hexanucleotides and that the aminosugar of the antibiotic is situated in the minor groove of the hexamer duplex, partly eclipsing the third base pair. A quantitative determination of the complexation of DAU with deoxyhexanucleotides has been made using the experimantal concentration and temperature dependences of the drug proton chemical shifts, which have beenanalysed in terms of the equilibrium reaction constants, limiting proton chemical shifts and thermodynamical parameters of different drug-DNA complexes (1:1, 1:2, 2:1, 2:2) in aqueous solutions. It is found that there is an apparent site-specificiety on drug binding with a triplet nucleotide sequence containing two adjacent C.G base pairs flanked by an A.T base pair in the 5`-d(CGTACG) hexamer over a triplet with three succesive C.G base pairs as in 5`-d(CGCGCG). The major reason for the preferential binding of DAU is found to stem from hydrophobic interactions of its aminosugar moiety in the minor groove of the double helix. The most favourable structure of the 1:2 complexes have been derived from the induced limiting proton chemical shifts of the drug in the intercalated complexes with the hexanucleotide duplex, in conjunction with 2D NOE data, which are in good agreement with X-ray results.