A novel fluorescent benzo[g]imidazo[4,5-c]quinoline nucleoside (BIQ)A (1) comprising a 3-deaza-2'-deoxyadenosine skeleton was developed and used to monitor (BIQ)A-C base-pair formation in oligodeoxynucleotide (ODN) duplexes. The newly synthesized (BIQ)A exhibited distinct photophysical properties associated with its protonated/deprotonated forms (monomer: pKa 6.2) via dramatic changes in its absorption and fluorescence spectra. In ODN duplexes, the induced protonation of (BIQ)A occurred, even under alkalescent conditions when cytosine was the opposite base on the complementary strand; the resulting (BIQ)A-C base pairs were stable. By monitoring the protonation of (BIQ)A under neutral and alkalescent conditions, we could clearly discriminate cytosine through spectral changes in absorption and fluorescence. Similarly, we found that the demonstrated 3-deaza-2'-deoxyadenosine (3z)A forms a stable base pair with cytosine via N(1) protonation in ODN duplexes under neutral and acidic conditions (pH < 7.0). At lower pH values, (3z)A-containing ODNs could clearly discriminate cytosine through melting temperature (Tm) measurements. Therefore, ODN probes containing indicator nucleosides, such as (BIQ)A and (3z)A, exhibit great potential as bioprobes for genetic analysis and structural studies of nucleic acids.