3-Deazaguanine (3DG) and its 2-N-acylated derivatives were incorporated into 2'-O-methyl-RNAs, and the hybridization and base discrimination properties were clarified by measuring UV-melting profiles. The 2'-O-methyl-RNA incorporating a 3DG hybridized less strongly in comparison to that without 3-deaza modification to both fully-matched and singly mismatched target strands. The T(m) decrease was the largest in the case of the fully matched duplex so that the base discrimination ability was decreased. Interestingly, the acetylation of the amino group of 3DG could compensate the T(m) decrease of the fully matched duplex by 3DG. Moreover, it was also proved that the base pair of 2-N-acetyl-3-deazaguanine and uracil was much weaker than the G-U pair. These results indicate the possibility of the development of guanine analogs that can recognize cytosine more precisely than guanine by use of the 2-N-acyl-3-deazaguanine skeleton.