A potential alternate substrate for orotidine-5′-monophosphate decarboxylase, 2- thio-orotidine-5′-monophosphate, was synthesized enzymatically and purified by a modification of a previous account (K. Shostak, and M. E. Jones 1992, Biochemistry31, 12155–12161). Characterization of the product was confirmed by mass spectrometry, 31 P NMR, and utilization by orotate phosphoribosyltransferase in the direction of pyrophosphorolysis. The previous work probably did not result in the purification of the desired compound, as evidenced by our observation of 2-thioOMP's sensitivity to high temperature, as used previously. Using a very sensitive HPLC assay for the potential decarboxylated product 2-thioUMP, no measurable activity of ODCase toward the alternate substrate was observed, representing a decarboxylation rate decreased by 10 −7 from the k cat for ODCase toward OMP. Additionally, 2-thioOMP effects no inhibition of ODCase decarboxylation of OMP at a concentration of 50 μM, indicating a poor ability to bind to the ODCase active site. The results bear implications for proposed mechanisms for catalysis by ODCase.