Pyrimidines have many important roles in cellular physiology, as they are used in the formation of DNA, RNA, phospholipids, and pyrimidine sugars. The first rate-limiting step in the de novo pyrimidine synthesis pathway is catalyzed by the carbamoyl phosphate synthetase II (CPSase II) part of the multienzymatic complex Carbamoyl phosphate synthetase, Aspartate transcarbamoylase, Dihydroorotase (CAD). CAD gene induction is highly correlated to cell proliferation. Additionally, CAD is allosterically inhibited or activated by uridine triphosphate (UTP) or phosphoribosyl pyrophosphate (PRPP), respectively. The phosphorylation of CAD by PKA and ERK has been reported to modulate the response of CAD to allosteric modulators. While there has been much speculation on the identity of CAD phosphorylation sites, no definitive identification of in vivo CAD phosphorylation sites has been performed. Therefore, we sought to determine the specific CAD residues phosphorylated by ERK and PKA in intact cells. We observed the PKA-induced phosphorylation of Ser¹⁴⁰⁶ and Ser¹⁸⁵⁹ HEK-293 cells. Surprisingly, while ERK phosphorylated CAD on multiple residues in vitro, CAD was not an ERK substrate in HEK-293 cells. We determined the identity of a previously unknown phosphopeptide in CAD isolated from HEK-293 cells. We also observed that the phosphorylation of CAD in HEK-293 cells is important for the maintenance of CPSase II activity. In addition to investigating the regulation of CAD by phosphorylation, we have identified a novel protein-protein interaction between CAD and the human cell cycle checkpoint protein hRad9. The interaction was mapped to the CPSase II portion of CAD, and the binding of hRad9 to CAD induced a significant activation of CPSase II activity. Taken together, these studies demonstrate novel mechanisms of CAD regulation in mammalian cells.