A previously unknown 5'-nucleotidase (5'-ribonucleotide phosphohydrolase, EG 126.96.36.199) (5'-Nase) specific for orotidine 5'-monophosphate (OMP) has been discovered. This enzyme, orotidine 5'-monophosphate phosphohydrolase (OMPase), was isolated from mouse liver microsomes as a separate entity from the nonspecific 5'-Nase. OMPase was partially purified and is shown to cleave OMP to orotidine and inorganic phosphate. The enzyme has negligible activity towards UMP, CMP, dTMP, AMP, IMP, GMP, XMP, 6-azauridine 5'-monophosphate, 1-β-D-ribofuranosylbarbituric acid 5'-monophosphate (BMP), 2'-UMP, 3'-UMP, 2'-AMP, 3'-AMP, ribose 5-phosphate, and β-glycerophosphate, all of which - with the exception of the 2'- or 3'-monophosphates, ribose 5'-phosphate, and β-glycerophosphate - are substrates for 5'-Nase. Both enzymes are inhibited by NaF, but only OMPase is inhibited by SH reagents. OMPase is not inhibited by orotidine, orotate, BMP, concanavalin A, or tetramisole (an alkaline phosphatase inhibitor). OMPase has a M(r) 53,000, K(m) value of 1 mM for OMP, and V(max) value of 49 nmol/min.mg of protein at the present stage of purification. OMPase activity has also been detected in various mammalian tissues including normal human tissues, human tumor xenografts, lymphocytes, and rat liver. OMPase may be responsible, in part, for the low levels of intracellular 'free' OMP and for orotidine accumulation in cells treated with 6-azauridine and patients suffering from orotic aciduria.