Synthetic oligonucleotides have been proposed as a new rationally designed class of pharmaceuticals with a mechanism of action based upon a Watson-Crick and/or Hoogsteen type of base pairing with RNA or DNA regions of interest. Two series of 3'-cholesterol and/or 5'-cholesterol conjugated oligonucleotides have been synthesized. The primary structure of these compounds was conceived in a way that should allow a hydrophobic interaction to take place upon bringing the cholesteryl moieties into proximity via a hybridization event. In the first group of compounds the cholesteryl group was tethered to the opposite ends of two oligonucleotides, tandemly addressed to the same complementary strand. An increase in the Tm of duplexes up to 13.3 degrees C was observed in comparison to unmodified oligomers. We observed a higher level of mismatch discrimination for the two contiguous oligonucleotide cholesterol conjugates compared to one continuous oligomer of the same length. A second group of compounds was synthesized as 5',3'-bis-cholesterol containing oligomers, capable of forming 'clamp-shaped' triple-stranded complexes, where cholesterol groups were attached to the termini of duplex and triplex forming domains. Stabilization of triplexes by up to 30 degrees C due to inter-cholesteryl interaction was observed. We detected no triplex formation with a mismatched target. These data suggest that significant stabilization of complexes of nucleic acids could be achieved through inter-cholesteryl hydrophobic interaction.