Cholic acid and its deoxy derivatives were found to increase the melting point of oligonucleotide duplexes when coupled to their 5'-termini. For duplexes of mixed-sequence octamers, the melting point was 8-11 degrees C higher with the steroid appendage than without. For the self-complementary hexamer TGCGCA, a 21 degrees C melting point increase was measured in the presence of steroid appendage and -18 kcal/mol in Delta Delta H degrees. The affinity increases were accompanied by increased discrimination against mismatches at the two terminal base pairs and increased hypochromicity, as well as an improved DNA/RNA discrimination for the non selcomplementary sequence. Cross-peaks in the NOESY spectrum of (chl-T*GCGCA)(2), where chi denotes the cholic acid residue and T* a 5'-amino-5'-deoxythymidine residue, point toward stacking interactions in which the methyl groups of the steroid face the nucleobases. Our results indicate that bile acids can form a specific complex with terminal T:A base pairs of double-stranded DNA.