Publisher Summary This chapter describes the activity, specificity and structural chemistry of glutamate carboxypeptidase. Glutamate carboxypeptidases are characterized as Zn2+ requiring exopeptidases with specificity for glutamate and this specificity is not absolute as carboxypeptidase G was reported as exhibiting activity against pteroyl compounds with C-terminal aspartic acid. Physicochemical properties and substrate affinities for folic acid, and a variety of analogs are compared for the G1 and G2 enzymes. Carboxypeptidase G2 is a dimeric protein of 83,600 kDa containing two atoms of zinc per subunit. There are no disulfide bonds and the complete nucleotide and amino acid sequences were resolved following cloning of the gene into Escherichia coli. In both native and recombinant form the enzyme is located in the periplasmic space, targeted by a 22 amino acid signal peptide. Carboxypeptidase G2 has been crystallized and the crystal structure determined at 2.5 A resolutions. Each subunit of the molecular dimer consists of a larger catalytic domain containing two zinc ions at the active site and a separate smaller domain that forms the dimer interface.