Systematic kinetic, equilibrium binding, melting, and electrophoretic studies were carried out with oligonucleotides to determine the sequence specificities of chromomycin A3 (CHR) binding to DNA at the self-complementary tetranucleotide level. Decamers of the forms d(GTA-XGCY-TAC) and d(GTA-XCGY-TAC), with X, Y = A, G, C, or T, were used for this purpose. Results indicate that the binding preferences for CHR are in the order -GGCC- > -CGCG- > -GCGC-, -CCGG- > -AGCT- > -ACGT-, -TGCA- > -TCGA-. Detergent-induced drug dissociation studies revealed that CHR dissociates very slowly from both -GGCC- and -CGCG- sequences, with the former being measurably slower than the latter which in turn is at least an order of magnitude slower than the rest of the sequences. Thermal denaturation measurements indicate that the binding of CHR stabilizes the DNA duplex, with -GGCC- and -CGCG- exhibiting the largest effects. Results of gel electrophoretic retardation experiments support our general findings on the relative binding order. Our experimental results support earlier NMR findings by other researchers implicating the preference of aureolic acid drugs at the 5'GC3' step and further reveal significant modulations by the adjacent base pairs. Attempts were made to rationalize our results with the known detailed structural information from NMR studies.