Hepatitis delta virus ribozymes have been proposed to perform self-cleavage via a general acid/base mechanism involving an active-site cytosine, based on evidence from both a crystal structure of the cleavage product and kinetic measurements. To determine whether this cytosine (C75) in the genomic ribozyme has an altered pK(a) consistent with its role as a general acid or base, we used (13)C NMR to determine its microscopic pK(a) in the product form of the ribozyme. The measured pK(a) is moderately shifted from that of a free nucleoside or a base-paired cytosine and has the same divalent metal ion dependence as the apparent reaction pK(a)'s measured kinetically. However, under all conditions tested, the microscopic pK(a) is lower than the apparent reaction pK(a), supporting a model in which C75 is deprotonated in the product form of the ribozyme at physiological pH. While additional results suggest that the pK(a) is not shifted in the reactant state of the ribozyme, these data cannot rule out elevation of the C75 pK(a) in an intermediate state of the transesterification reaction.