DNA binding modes of distamycin (DST) were investigated via comparative binding studies with oligomeric duplexes of the form d(GCG-X-GCG).d(CGC-Y-CGC), where Y is complementary to X and X = 4- or 5-base binding site. It was found that 1:1 and 2:1 drug-duplex complexes exhibit distinctly different circular dichroic (CD) spectral characteristics and can, thus, serve as diagnostic tools for binding mode differentiation. CD intensity profiles at 265 or 275 nm as a function of drug to DNA ratios can reveal the extent of binding cooperativity for 2:1 complex formation (i.e., the relative binding affinities of 2:1 vs 1:1) at a 5-base-paired binding site. Comparison of these profiles leads to the following qualitative ranking for the binding cooperativity for the studied sites: AAGTT, ATATA >/= AAACT > AATAA, AAATA, AAAGT > AATAT > TAAAA >/= AAATT >/= AAAAA >/= ATAAA, AAAAT. The plausibility of this ordering is strengthened by its agreement with the ranking established by earlier NMR studies on some of the sequences. The significantly slower DST dissociation kinetics of the 2:1 complexes as compared to those of 1:1 made the kinetic measurements of SDS-induced dissociation by the stopped-flow technique possible. The results indicate that the AAGTT site exhibits the slowest DST dissociation rate, with a characteristic time of 35 s. The rates of dissociation in general correlate reasonably well with the cooperativity order found via equilibrium CD measurements (the higher the binding cooperativity, the slower the rate of dissociation). Base sequence specific binding of DST was also found for the 1:1 complex formation at the 4-base-paired sites, with AAAA, TTTT, ATTT, and AAAT sequences exhibiting the highest binding affinities.