The reversible binding of neocarzinostatin chromophore to polynucleotides was studied in order to understand the molecular basis of its base sequence-specificity in DNA damage production. Studies of the spectroscopic and thermodynamic properties of chromophore-polynucleotide interactions reveal that the binding of the chromophore to poly(dA-dT) is qualitatively and quantitatively different from that to poly(dG-dC) (and poly(dI-dC]. From these and other experiments using double-stranded mixtures of homopolynucleotides, it is proposed that the observed A T specific intercalation might result from differential recognition of minor variations in the B-DNA type structure by the chromophore.