Eighty pyrimidine base analogs were evaluated as inhibitors of mouse liver orotate phosphoribosyltransferase (OPRTase, EC 126.96.36.199). Based on these findings and an extensive literature review, a structure-activity relationship has been formulated for the binding of pyrimidine base analogs to OPRTase. This study provides a basis for the rational design of new inhibitors of this enzyme, and several such compounds are proposed. Additionally, 4,6-dihydroxypyrimidine has been found to be a potent OPRTase inhibitor. Eleven OPRTase inhibitors were also evaluated as inhibitors of orotidine 5′-monophosphate decarboxylase (ODCase, EC 188.8.131.52). 5-Azauracil, 5-azaorotate, and barbituric acid inhibited ODCase significantly only after preincubation with PRPP and MgCl2 in the presence of cytosol. © 1984.