The pyrG gene encoding cytidine 5'-triphosphate (CTP) synthetase which catalyzes the terminal reaction for de novo synthesis of pyrimidine nucleotides was cloned and sequenced from Mycobacterium bovis BCG. The BCG pyrG was screened in lambda ZAPII library employing a DNA probe which was constructed from PCR based on a consensus pattern of glutamine amidotransferase type I. The BCG CTP synthetase deduced from a nucleotide sequence consisting of 586 amino acid residues with a calculated molecular weight of 63,642 daltons. Alignment of BCG CTP synthetase with all sequences available in GenBank indicates that the residues suggested as a catalytic triad for glutamine hydrolysis at the carboxyl terminal domain and an amino terminal segment are completely conserved among all CTP synthetases.