2-Acylamino-1,8-naphthyridine (1), which possesses hydrogen bonding groups fully complementary to guanine (G), selectively binds to a single G bulge of duplex DNA. The melting temperature (Tm) of the duplex containing a G bulge was increased by the presence of 1, whereas no increase of Tm was observed for the duplexes containing adenine (A) and thymine (T) bulges as well as for normal duplex. Riboflavin-sensitized photooxidation of DNA containing GG steps opposite to G and A bulges was selectively inhibited by the presence of 1 at the G bulge. DNase I footprinting titration indicated a selective binding of 1 to the G bulge with an association constant of 3.4 ± 1 × 104 M-1. In the presence of 1, CD spectra of the G bulge-containing duplex noticeably changed, being accompanied by the induced CD at 300−350 nm, whereas no CD spectral change was observed for the duplex containing A bulge. Both the hydrogen bonding groups complementary to G and the planar bicyclic ring system are essential for the complex formation between G bulge and 1.