Folate biochemical pathway enzymes such as folylpolyglutamate synthetase (FPGS) are key elements in the folate pathway. The role of FPGS is to add glutamate residues to folates and antifolates, trapping them in the cell and increasing their affinity for subsequent enzymatic reactions. FPGS may also be an indicator of response to both clinically established and novel antifolate drugs such as pemetrexed; knowledge of their level of expression in tumors may enable their optimal use by identifying potentially responsive subgroups of patients. In spite of its key role in both nucleotide biosynthesis and possible role as a determinant of response in chemotherapy, monoclonal antibodies to FPGS suitable for immunohistochemical analysis of formalin fixed and paraffin embedded biopsy samples, or that can be used for Western blot analysis, are not commercially available. The aim of this study was to generate a monoclonal antibody that could be used to detect specific expression of FPGS in paraffin embedded tissues. A 228 amino acid region of the FPGS sequence was expressed as a recombinant fusion protein and used as an antigen to generate monoclonal antibodies. ELISA and Western blot studies identified specific reactivity of the NN3.2 antibody to the recombinant protein and a single 60 kDa protein in whole cell lysates from cell lines known to express FPGS. Immunohistochemical analysis of FPGS using hybridoma clone NN3.2 in a panel of normal tissues demonstrated wide expression including strong immunoreactivity in the brush border and crypts of colon, liver hepatocytes, and lymphoid cells. Analysis of a panel of malignant and benign tissues demonstrated wide expression with variable intensities of staining and patterns of cytoplasmic reactivity. Stronger staining was observed in malignant tissue compared with that of normal adjacent tissue, particularly in ovarian and colon adenocarcinoma cases. Our results show that clone NN3.2 is a sensitive tool for detection of FPGS in paraffin-embedded tissues.