Folate biochemical pathway components such as FR-alpha are determinants of response to novel antifolate drugs such as pemetrexed. Knowledge of their level of expression in tumors will enable their optimal use by identifying potentially responsive subgroups of patients. In spite of its importance in the diagnosis and treatment of cancer, monoclonal antibodies to FR-alpha suitable for immunohistochemical analysis of formalin-fixed and paraffin-embedded biopsy samples, or that can be used for Western blot analysis, are not available. The aim of this study was thus to generate a monoclonal antibody that could be used to detect specific expression of FR-alpha in paraffin-embedded tissues. A 189 amino acid region of the FR-alpha sequence was expressed as a recombinant fusion protein and used as antigen to generate monoclonal antibodies. Studies by ELISA and Western blot identified specific reactivity of the BN3.2 antibody to the recombinant protein and a single 40 kD protein in whole cell lysates from cell lines known to express FR-alpha. Immunohistochemical analysis of FR-alpha using hybridoma clone BN3.2 in a panel of normal tissues demonstrated expression limited to ovarian epithelia, placental trophoblasts, and proximal kidney tubules. Analysis of a panel of malignant and benign tissues demonstrated limited expression with variable intensities of staining and patterns of both membrane and cytoplasmic reactivity observed between cases. In the majority of malignant ovarian tumors, high intensity staining was observed, predominantly localized to the plasma membrane. Our results show that clone BN3.2 is a sensitive tool for detection of FR-alpha in paraffin-embedded tissues. This preliminary study also supports its use in immunohistochemical studies to determine the role of FR-alpha as a tumor prognostic marker and a possible therapeutic target.