Complex U, which contains the last two enzymes (orotate phosphoribosyltransferase (EC 2.4.2.10) and orotidylate decarboxylase (EC 4.1.23)) of the six enzymes for the de novo biosynthesis of UMP, has been purified 200-fold from mouse Ehrlich ascites cells. The specific activity of the orotate phosphoribosyltransferase and the orotidylate decarboxylase activities of the complex were 0.115 and 0.290 mumol of product/mg of protein/min; the recovery of the activities was high being 20 to 30%. The rate of the two activities remained similar to that of the homogenate. At the sixth step of the fractionation, one can obtain a fraction that has lost phosphoribosyltransferase activity but retains decarboxylase activity. The apparent molecular weights, as determined by density gradient centrifugation, of the native complex and the fraction containing only decarboxylase activity are identical, 55,700 +/- 4,000. Both activities of complex U are labile to very mild treatments such as dilution, dialysis, or storage at 3 degrees. Dithiothreitol and 5-phosphoribosyl-1-pyrophosphate (PP-Rib-P), but not orotic acid or MgCl2, can stabilize either or both of the enzyme activities. The degree of stabilization by three of these chemicals varies with the reagent(s) used, with the nature of the treatment, and with the concentration of Complex U. When PP-Rib-P, Mg2+ and dithiothreitol are present in the diluting buffer the activity losses were slowed and then followed by a partial recovery of the phosphoribosyltransferase activity. Maximum activities of both enzymes are observed by adding undiluted complex to a complete reaction mixture without preincubation. The complex cannot be exposed to pH values of 4 or below, or pH 9 or above. The stability studies have led to the development of conditions that permit one, for the first time, to subject the complex to electrophoresis and to recover a large percentage of both enzyme activities, rather than only decarboxylase activity as has occurred in the past. The electrophoretic studies indicate that PP-Rib-P produces a complex whose conformation and/or net charge differ significantly from that of the complex in the absence of PP-Rib-P. Kinetic characteristics of the transferase are a pH optimum between 6.5 and 7.5, apparent Km values for orotate, PP-Rib-P, and Mg2+ of 1.9 muM, 16 muM, and 2.9 mM, respectively; for the decarboxylase, a sharp pH optimum of 7.0 is observed, and a Km value for orotidine 5'-phosphate of 0.8 muM.