An efficient procedure was developed to prepare homo-C-nucleosides. The β-allyl C-glycoside of D-ribose was transformed into the thienopyrimidine nucleoside and the benzodiazepine nucleoside of 2-deoxy-D-ribose. For incorporation into oligonucleotides by solid-phase synthesis, both derivatives were transformed into the related 3′-phosphoramidite building blocks. These phosphoramidites were then site-specifically incorporated into DNA oligonucleotides. The modified DNA strands were hybridized with different DNA and RNA strands, and their melting temperatures and circular dichroism spectra were studied. Interestingly, although in the case of DNA-DNA hybrids the nonnatural nucleosides caused a marked decrease in melting temperature, to levels even lower than those for mismatched hybrids, in the case of DNA-RNA hybrids very similar melting temperatures were measured for the nonnatural nucleosides and the unmodified oligonucleotides. 3′-Phosphoramidites of thienopyrimidine and benzodiazepine homo-C-nucleosides were prepared and site-specifically incorporated into DNA. These modified oligonucleotides were hybridized with different DNA and RNA strands in order to study their duplex characteristics in terms of their melting temperatures and circular dichroism spectra.