Two carbamyl phosphate synthetases, the first an arginine-synthetic enzyme (CPS(arg)) and the second a pyrimidine-synthetic enzyme (CPS(pyr)), are shown to be present in Neurospora. The two enzymes can be separated on the basis of size and are distinguished by several different properties. Both CPS(pyr) and CPS(arg) have substrate requirements of adenosine triphosphate, HCO(3) (-), and l-glutamine, although NH(4) (+) in high concentration will partially replace glutamine. CPS(pyr) activity can be completely inhibited by 5 x 10(-4) to 10 x 10(-4)m uridine triphosphate (UTP). CPS(pyr) is cold-labile and can be protected against cold inactivation by UTP. The synthesis of CPS(pyr) and aspartate transcarbamylase (ATC), the initial enzymatic steps of the pyrimidine pathway, are co-derepressed by pyrimidine starvation. Mutations affecting CPS(pyr) and ATC all map at the same locus, pyr-3. Three classes of mutants with respect to the two activities were found: CPS(+)ATC(-), CPS(-)ATC(+), and CPS(-)ATC(-). The distribution of these mutants on the genetic map, together with other data, indicate that the two activities are carried by a bifunctional protein.