At high salt concentrations, ethidium bromide (EB) is shown to form a fluorescent complex with helical polynucleotides which has a strikingly enhanced fluorescence quantum yield. In this paper we present evidence that formation of this complex (complex I) ia specific for base-paired regions either in DNA, RNA or RNA: DNA hybrids, and that the basis of this specificity is intercalation of the dye between base pairs. At low salt concentrations a second mode of binding EB ‡ (as complex II) is observed: it appears to be electrostatic binding of the cationic dye to negatively charged phosphate groups. Complex II can be distinguished readily from complex I by its fluorescence properties. The nature of the DNA binding sites for EB has been investigated by: (a) measurement of their number, and dependence of the binding on pH and salt concentration, as well as on the secondary structure and GC content of the DNA; (b) study of the energy transfer from DNA to EB; (c) studies of competition for DNA between EB and Na +, Mg 2+, quinacrine and actinomycin, and (d) characterization of complex I by flow dichroism, thermal melting curves and buoyant density.