The spin-labeled nucleoside C-T, containing 2,2,6,6-tetramethyipiperidine-l-oxyL (TEMPO) conjugated to the exocyclic amino group of C, was used to detect single-base mismatches in duplex DNA for the first time by electron paramagnetic resonance (EPR) spectroscopy. Furthermore, the EPR spectra of the fully base-paired duplex (TC-G) and the mismatches (C-T-A, C-T-C, and C-T-T) were significantly different, showing that the probe can identify its base-pairing partner in DNA. At lower pH, the mobility of C-T-A, TC.C, and C-T-T became higher, consistent with increased protonation of the mismatched pairs. Although the duplexes for each of the three flanking sequences tested gave distinguishable EPR signals, the best discrimination between base pairs was achieved for sequences containing a flanking A-T base pair, in particular 5'-d(G(T)CA) and 5'-d(IICA). This study shows that minor structural variations in nucleic acids can be detected with carefully chosen spin [abets in conjunction with EPR spectroscopy.