Carbamoyl phosphate synthetase II (CPSII) is part of carbamoyl phosphate synthetase/aspartate transcarbamoylase/dihydroorotase (CAD), a multienzymatic protein required for the de novo synthesis of pyrimidine nucleotides and cell growth. Herein, we identify CAD as a substrate for caspase-3 degradation in both in vitro and in vivo models of apoptosis. Withdrawal of interleukin-3 or incubation with staurosporine (STS) or doxorubicin (Dox) resulted in proteolytic cleavage of CAD in a myeloid precursor cell line (32D) or in a cell line over-expressing CAD. The rapid decline in the CPSII activity paralleled the degradation of CAD and preceded the appearance of Annexin-V-stained apoptotic cells and DNA fragmentation. These events correlated closely with the activation of caspase-3 in these cells and were prevented by the cell-permeable caspase inhibitor N-benzyloxycarbonyl-Asp-Glu-Val-Asp fluoromethyl ketone. Moreover, the incubation of purified CAD with recombinant caspase-3 in vitro generated CAD fragments that were similar to those obtained in vivo. Edman sequencing revealed that two of the major caspase-3 cleavage sites occurred at the sequences EAVD/G and VACD/G within the catalytic (B2) and allosteric (B3) domains of CAD, thus providing a potential mechanism for the rapid inactivation of CPSII during apoptosis. Consistent with this, an enhanced loss of the intracellular pyrimidines (UTP and CTP) was observed in response to STS or DOX-induced apoptosis. Therefore, these studies show that CAD is a novel target for caspase-dependent regulation during apoptosis and suggest that the selective inactivation of pyrimidine nucleotide synthesis accompanies the process of apoptosis.