Fluorescent Hoechst 33258 analogues have been synthesized in which the terminal phenol moiety is employed as a site for the introduction of a linker to permit covalent attachment of the fluorophores to oligo(deoxynucleotides). Hybridization by the DNA-Hoechst conjugates to target sequences generates the DNA minor groove structure and triggers a binding event by the tethered Hoechst agent. The attendant fluorescence signal reports upon this hybridization event. Conjugation of the Hoechst derivatives to the DNA sequences employs a cystamine linker tethered to an internucleotide phosphorus residue. This mode of conjugation maximizes the versatility of linker placement and minimizes the associated chemistry required to introduce the linker. Two related Hoechst derivatives have been synthesized; both contain a bromoacetamido linker for conjugation to the oligonucleotides. With each Hoechst derivative, two pairs of diastereomeric (R(p) and S-p) oligo(deoxynucleotide) conjugates were prepared to provide the tethered Hoechst groove binders with two different orientations within the dA-dT rich minor groove. T-m measurements suggest that while all of the conjugates provide some increased duplex stability, the diastereomeric conjugates tentatively assigned the R(p) configuration exhibit nearly 20 degrees C increases in T-m values for the double-stranded dodecameric complexes, while those tentatively assigned as the S-p diastereomers exhibit only moderate 4-8 degrees C increases in T-m values. The fluorescence characteristics of the conjugates are more variable, with one complex exhibiting a 23-fold enhancement in quantum yield effects, very similar to that observed for a free untethered Hoechst 33258 fluorophore bound to duplex DNA.