A new method is presented for the synthesis of oligonucleotides containing 15N-enriched 5-fluorocytosine (FC). Due to the reduced pK of FC, the amino protons of an unpaired FC residue may be observed at lower values of solution pH. The labeled FC residue has been placed as a template base at a model DNA replication fork. The amino protons of the FC residue have been identified in isotope-edited NMR spectra. Data is presented for a template FC residue unpaired, paired with guanine, and mispaired with adenine. These studies demonstrate the utility of labeled FC in examining unusual DNA structures.