Tethering the Hoechst 33258 fluorophore to the 5’-terminus of the polypyrimidine third strand of parallel-stranded DNA triplexes results in complexes with increased T-m, values that vary with both the pH of the solution and the sequence of the target DNA. These pH/sequence effects are Likely related to the presence of M+-G-C base triplets, which can result in either charge-charge effects involving the base triplet and the charged piperazine ring of the ligand or changes in the nature of the minor groove binding site resulting from the introduction of the N-2-exocyclic amino group(s). As with DNA duplexes, sequence targets rich in A-T base pairs are most effective in taking advantage of such ligand-induced stability. A duplex sequence rich in A-T base pairs adjacent to the triple helix also appears to permit adjacent ligand binding; that is, the tripler is stabilized by the binding of the tethered minor groove ligand in an A-T rich duplex adjacent to the site of the tripler. The Hoechst ligand can be very effective in stabilizing G-T-A base triplets which are generally less effective in tripler formation, presumably as the result of a single interstrand G-T hydrogen bond. Stabilization may occur in part because the (AATT)(2) minor groove may offer the ligand a preferred binding site as has been documented for this sequence in DNA duplexes. Binding to the tripler results in an enhanced quantum yield for the fluorophore, the magnitude of which is dependent upon sequence effects. Stopped flow experiments have provided some insight into the nature of the process; rapid ligand binding to the duplex target is followed by a slower process, one interpreted to reflect the third strand binding to generate the conjugated DNA tripler. Although not conclusive, the experiments suggest that the Hoechst conjugated polypyrimidine strands bind to the target duplex by two simultaneous sets of interactions : (i) third strand binding in the major groove and (ii) tethered Hoechst 33258 ligand binding in the minor groove.