AbstractGlutamate carboxypeptidase II (EC 3.4.17.21) catalyzes the hydrolysis (Km = 0.2 μM) of the neuropeptide N‐acetylaspartylglutamate to yield N‐acetylaspartate and glutamate and also serves as a high‐affinity folate hydrolase in the gut, cleaving the polyglutamate chain to permit the absorption of folate. N‐acetylaspartylglutamate is an agonist at the mGluR3 metabotropic receptor and a source of extracellular glutamate through hydrolysis by glutamate carboxypeptidase II. Given the important role of glutamate in brain development and function, we were interested in the effects of a null mutation of glutamate carboxypeptidase II that would potentiate the effects of N‐acetylaspartylglutamate. The PGK‐Neomycin cassette was inserted to delete exons 9 and 10, which we previously demonstrated encode for the zinc ligand domain essential for enzyme activity. Successful germline transmission was obtained from chimeras derived from embryonic stem cells with the targeted mutation of glutamate carboxypeptidase II. Homozygous null mutants did not survive beyond embryonic day 8. Folate supplementation of the heterozygous mothers did not rescue the homozygous embryos. Mice heterozygous for the null mutation appeared grossly normal and expressed both mutated and wild‐type mRNA but the activity of glutamate carboxypeptidase II is comparable to the wild‐type mice. The results indicate that the expression of glutamate carboxypeptidase II is upregulated when one allele is inactivated and that its activity is essential for early embryogenesis. Synapse 50:285–292, 2003. © 2003 Wiley‐Liss, Inc.