AbstractOne‐dimensional and two‐dimensional (2D) nmr experiments were carried out on an oligonucleotide duplex that contains an unpaired cytosine, d(GCGAACAAGCG)·d(CGCTTTTCGC), which will be referred to as the C‐bulge decamer. Evidence from one‐dimensional nuclear Overhauser effect (NOE) experiments on the exchangeable protons indicates that the unpaired cytosine is extrahelical. This conclusion is also supported by numerous cross‐peaks in the 2D NOE spectroscopy (NOESY) spectrum of the nonexchangeable protons. The assignments for all of the resonances, with the exception of the H5′ and H5″ resonances, have been made through the use of 2D NOESY, correlated spectroscopy (COSY), and relayed COSY experiments. The temperature dependence of the C(H6) resonance chemical shifts indicates that the unpaired cytosine shows unusual behavior compared to other cytosines in the duplex. A comparison of chemical shifts for all, the assigned resonances of the duplexes with and without the unpaired cytosine suggests that the majority of the structural perturbation is localized in the A·T tract surrounding the unpaired base. The behavior of the imino resonances as a function of temperature also indicates that the perturbation to the duplex is localized and destabilizes the A·T base pairs adjacent to the unpaired base. © 1993 Joh