A highly purified enzyme complex (from bakers' yeast), which possesses carbamylphosphate synthetase (CPSase) and aspartate transcarbamylase (ATCase) activities and a regulatory site for both activities at which feedback inhibition is exerted by UTP, was subjected to heat at 50 °C, and all three of the above parameters were followed. A treatment of 5 min was sufficient to cause disaggregation of the complex (original molecular weight 600 000) and appearance of subunits of approximately one-quarter the size of the unheated material. These subunits possessed only the ATCase activity, which was insensitive to uridine triphosphate; no component possessing CPSase activity was found in the sucrose density gradient. The ATCase was relatively resistant to heat, but there was a rapid and roughly parallel decline in the CPSase and in the sensitivity of the ATCase to inhibition by UTP. However, the residual CPSase activity, at any duration of heating, remained fully sensitive to inhibition by uridine triphosphate. CPSase activity was optimal at temperatures between 25° and 30° whereas the ATCase followed the Arrhenius law to 37 °C. Two alternative hypotheses of the structure of the enzyme complex were put forward to account for these data. Both of these hypotheses require that CPSase activity be a function of the oligomeric aggregate and not of individual subunits.