The new ligand 6 is structurally related to ethidium bromide (EB) and other diazapyrenium compounds (1, 2) studied earlier, but exhibits significant differences. In contrast to ligands such as 1 and 2 the new 6 is stable over a large pH range. At concentrations > 10−4 mol dm−36 forms a dimer, which is characterised by NMR analysis. The association constants of 6 are larger with purine than with pyrimidine nucleotides and are the same for AMP, ADP or ATP, with an absence of any charge effects. Surprisingly, under conditions close to saturation of intercalation binding sites association of 6 with single-stranded poly U and poly A is 100 to 1000 times more efficient than that of EB or analogue 2. Also the affinity of 6 toward poly U is significantly higher than toward poly C. CD spectra indicate that stacking of 6 with poly U induces a strong helicity in the usually disordered structure of this single strand. The binding of 6 to poly C is pH dependent, as a consequence of the known formation of a poly CH+–poly CH+ double strand at pH < 5. With double-stranded polynucleotides the binding affinity of 6 and EB is similar for RNA homopolymers. Striking fluorescence differences, however, are observed with complexes of 6–poly GC (decrease of emission intensity of 6 by 80%), and 6–poly AU (increase by 100%). Similar effects are also observed for the DNA polynucleotides. At higher ligand to phosphate ratios (r > 0.2) 6 shows with double strands electrostatic binding in addition to intercalation, with ensuing opposite effects in fluorescence emission. The biphasic melting profiles of poly dA–poly dT with 6 differ sharply from those observed with 1 and EB and are in line with the dependence of binding modes obvious from the fluorescence studies. Melting analyses show with calf thymus DNA and with poly dA–poly dT an increase (ΔΔT) in melting temperature with 6 about twice as high as that with 1 or EB. With the RNA model poly A–poly U the ΔTm values of 6 at low ionic strength are about seven times higher than those observed with 2 or with EB. In contrast to other 4,9-diazapyrenium compounds which show with poly A–poly U a destabilising and a stabilising step, 6 gives only positive ΔT values at both pH = 5.0 and pH = 7.0. Extensive supporting data for this paper are available as supplementary material in electronic form, allowing re-evaluation of experimental results with suitable programs for curve fitting.