Chromomycin A3 (CHR) binding to the duplex d(CAAGTCTGGCCATCAGTC).d(GACTGATGGCCAGACTTG) has been studied using quantitative footprinting methods. Previous NMR studies indicated CHR binds as a dimer in the minor groove. Analysis of autoradiographic spot intensities derived from DNase I cleavage of the 18-mer in the presence of various amounts of CHR revealed that the drug binds as a dimer to the sequence 5'-TGGCCA-3',3'-ACCGGT-5' in the 18-mer with a binding constant of (2.7 +/- 1.4) x 10(7) M-1. Footprinting and fluorescence data indicate that the dimerization constant for the drug in solution is approximately 10(5) M-1. Since it has been suggested that CHR binding alters DNA to the A configuration, quantitative footprinting studies using dimethyl sulfate, which alkylates at N-7 of guanine in the major groove, were also carried out. Apparently, any drug-induced alteration in DNA structure does not affect cleavage by DMS enough to be observed by these experiments.