A series of benzothiazole-substituted trisbipyridine ruthenium(II) analogues {[Ru(bpy)₂(4,5′-bbtb)]²⁺, [Ru(bpy) ₂(5,5′-bbtb)]²⁺ and [Ru(bpy)₂(5-mbtb)] ²⁺ [bpy is 2,2′-bipyridine, bbtb is bis(benzothiazol-2-yl)-2, 2′-bipyridine, 5-mbtb is 5-(benzothiazol-2-yl),5′-methyl-2,2′- bipyridine]} have been prepared and compared with the complex [Ru(bpy) ₂(4,4′-bbtb)]²⁺ reported previously. From the UV-vis spectral studies, substitution at the 5-position of the bpy causes the ligand-centred transitions to occur at considerably lower energy than for those with the functionality at the 4-position, while at the same time causing the emission to be effectively quenched. However, substitution at the 4-position causes the metal-to-ligand charge transfer to occur at lower energies. Fluorescent intercalator displacement studies indicate that the doubly substituted complexes displace ethidium bromide from a range of oligonucleotides, with the greater preference shown for bulge and hairpin sequences by the Λ enantiomer. Since the complexes only show small variation in the UV-vis spectra on the introduction of calf thymus DNA and a small increase in fluorescence they do not appear to be intercalators, but appear to associate within one of the grooves. All of the reported bisbenzothiazole complexes show reasonable cytotoxicity against a range of human cancer cell lines.