Hypochlorous acid (HOCl) from activated neutrophils at sites of inflammation can react with and damage biological molecules, including nucleic acids. The reaction of HOCl with cytosine analogues can generate multiple products, including 5-chlorouracil (ClU). In this paper, we have constructed oligonucleotides containing ClU paired opposite guanine (ClU-G). Melting studies indicate that oligonucleotide duplexes containing the ClU-G mispair are substantially less stable than those containing a ClU-A base pair. The melting temperature of the ClU-G mispair is not experimentally distinguishable from that of a T-G pair. NMR studies indicate that the ClU-G base pair adopts a wobble geometry at neutral pH, similar to a T-G mispair. The exchangeable protons of the ClU-G mispair broaden rapidly with an increase in temperature, indicating that the ClU-G mispair is less stable and opens more easily than the surrounding adjacent base pairs. Unlike the ClU-A base pair studied previously [Theruvathu, J. A., et al. (2009) Biochemistry 48, 7539-7546], the ClU-G mispair undergoes a pH-dependent structural change, assuming an ionized base pair configuration that approximates a Watson-Crick base pair at higher pH. Ionization of ClU in a DNA template could promote mispair formation and mutation, in accord with previous studies on other 5-halouracil analogues. The electron-withdrawing 5-chloro substituent facilitates ionization of the ClU N3 proton, promoting mispair formation, but it also renders the glycosidic bond susceptible to base cleavage by DNA repair glycosylases.