The URA7 gene of Saccharomyces cerevisiae encodes CTP synthetase (EC 6.3.4.2) which catalyses the conversion of uridine 5'-triphosphate to cytidine 5'-triphosphate, the last step of the pyrimidine biosynthetic pathway. We have cloned and sequenced the URA7 gene. The coding region is 1710 bp long and the deduced protein sequence shows a strong degree of homology with bacterial and human CTP synthetases. Gene disruption shows that URA7 is not an essential gene: the level of the intracellular CTP pool is roughly the same in the deleted and the wild-type strains, suggesting that an alternative pathway for CTP synthesis exists in yeast. This could involve either a divergent duplicated gene or a different route beginning with the amination of uridine mono- or diphosphate.