GMP synthetase (EC 6.3.4.1), a glutamine amidotransferase encoded by the guaA gene, catalyzes the synthesis of GMP from XMP. The guaA gene was subcloned from the Clarke and Carbon plasmid pLC34-10, and its nucleotide sequence was determined. The structural gene encodes a protein of 525 amino acid residues having a calculated molecular weight of 58,604. The amino acid sequence of the NH2 terminus of GMP synthetase was determined to verify the translation start site from the DNA sequence, showing the initiator methionine as the NH2-terminal residue instead of the previously reported arginine, and a 51-amino acid residue peptide was identified. A 68-base pair intercistronic region separates guaA from the upstream guaB gene in the polycistronic guaBA operon. The 3' end of the guaA mRNA, determined by S1 nuclease mapping, is 36-37 nucleotides downstream of the translation stop codon within a region of dyad symmetry that resembles a ρ-independent transcription termination site. A region of dyad symmetry with potential secondary structure was found in the intercistronic region.