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The pyrGgene of Lactococcus lactissubsp. cremoris, encoding CTP synthase, has been cloned and sequenced. It is flanked upstream by an open reading frame showing homology to several aminotransferases and downstream by an open reading frame of unknown function. L. lactisstrains harboring disrupted pyrGalleles were constructed. These mutants required cytidine for growth, proving that in L. lactis, the pyrGproduct is the only enzyme responsible for the amination of UTP to CTP. In contrast to the situation in Escherichia coli, an L. lactis pyrGmutant could be constructed in the presence of a functionalcddgene encoding cytidine deaminase. A characterization of the enzyme revealed similar properties as found for CTP synthases from other organisms. However, unlike the majority of CTP synthases the lactococcal enzyme can convert dUTP to dCTP, although a half saturation concentration of 0.6 mmfor dUTP makes it unlikely that this reaction plays a significant physiological role. As for other CTP synthases, the oligomeric structure of the lactococcal enzyme was found to be a tetramer, but unlike most of the other previously characterized enzymes, the tetramer was very stable even at dilute enzyme concentrations.