The mechanism of orotidine 5'-monophosphate decarboxylase was studied computationally by using the decarboxylation of orotic acid analogues as model systems. These calculations indicate that mechanisms involving proton transfer to the 2-oxygen or the 4-oxygen are energetically favorable, as compared to direct decarboxylation without proton transfer, for a series of model compounds where N1 is substituted with respectively H, CH3, and a tetrahrydrofuran moiety. Proton transfer to the 4-oxygen during decarboxylation is found to be energetically more favorable than 2-protonation, which is attributable to both the 4-oxygen site being more basic and an apparent intrinsic preference for the 4-protonation pathway. N-15 isotope effect calculations were also conducted, and compared to experimental 15N isotope effects previously measured at Nl by Rishavy and Cleland (Biochemistry 2000, 39, 4569-4574). The theoretical isotope effects establish, for the first time, that the experimental 15N isotope effect is consistent with decarboxylation without protonation, as well as with decarboxylation with protonation, at either O2 or at O4. Further-more, we propose herein an isotope measurement that could potentially distinguish among mechanisms involving protonation from those that do not involve proton transfer.