Hybridization thermodynamics were compared for oligonucleotide sequences containing 2'-fluoro dA, 2'-O-methyl A, 2'-O-ethyl A, 2'-O-propyl A, 2'-O-butyl A, 2'-O-pentyl A, 2'-O-nonyl A, 2'-O-allyl A, and 2'-O-benzyl A in place of deoxyadenosine. Although the effect of 2'-modified adenosine on duplex stability is sequence dependent, a clear trend is apparent. For six sequences containing a few 2'-modified adenosines in a background of unmodified deoxynucleotides, the average delta TM per substitution ranged from +1.3 degrees C for 2'-fluoro dA to -2.0 degrees C for 2'-O-nonyl A. For the 2'-O-alkyl series, the average delta TM per substitution correlates well with size of the substituent; the order of stability is 2'-O-methyl A > 2'-O-ethyl A > 2'-O-propyl A > 2'-O-butyl A > 2'-O-pentyl A > 2'-O-nonyl A. This correlation also extends to 2'-fluoro dA, 2'-O-allyl A, and 2'-O-benzyl A if chain length is measured by number of carbon atoms. When examined in the background of 2'-O-methyl ribonucleotides, all 2'-modified adenosines with a substituent no larger than 2'-O-pentyl stabilized the duplex nearly 2 degrees C per substitution compared to unmodified dA. These thermodynamic results and CD spectra of modified and unmodified hybrids support a model of DNA:RNA hybrids in which the geometry is between that of B-form and A-form.