The gas-phase substitution reactions of methyl chloride and 1,3-dimethyluracil (at the N1-CH(3)) are examined computationally and experimentally. It is found that, although hydrochloric acid and 3-methyluracil are similar in acidity, the leaving group abilities of chloride and N1-deprotonated 3-methyluracil are not: chloride is a slightly better leaving group. The reason for this difference is most likely related to the electron delocalization in the N1-deprotonated 3-methyluracil anion, which we explore further herein. The leaving group ability of the N1-deprotonated 3-methyluracil anion relative to the N1-deprotonated 3-methylthymine anion is also examined in the context of an enzymatic reaction that cleaves uracil but not thymine from DNA.