Human Prostate Specific Membrane Antigen (PSMA), also known as folate hydrolase I (FOLH1), is a 750-amino acid type II membrane glycoprotein, which is primarily expressed in normal human prostate epithelium and is upregulated in prostate cancer, including metastatic disease. We have cloned and sequenced the mouse homolog of PSMA, which we have termed Folh1, and have found that it is not expressed in the mouse prostate, but primarily in the brain and kidney. We have demonstrated that Folh1, like its human counterpart, is a glutamate-preferring carboxypeptidase, which has at least two enzymatic activities: (1) N-acetylated alpha-linked L-amino dipeptidase (NAALADase), an enzyme involved in regulation of excitatory signaling in the brain, and (2) a gamma-glutamyl carboxypeptidase (folate hydrolase). The 2,256-nt open reading frame of Folh1 encodes for a 752-amino acid protein, with 86% identity and 91% similarity to the human PSMA amino acid sequence. Cells transfected with Folh1 gained both NAALADase and folate hydrolase activities. Examination of tissues for NAALADase activity correlated with the mRNA expression pattern for Folh1. Fluorescent in situ hybridization (FISH) revealed Folh1 maps to only one locus in the mouse genome, Chromosome 7D1-2.