Pituitary adenomas cause significant morbidity caused by compression of regional structures or the inappropriate expression of pituitary hormones. However, little is known about the molecular changes that contribute to the development of these tumors. To investigate these changes, we recently used cDNA microarray analysis to identify several genes with altered expression patterns in pituitary adenomas. The folate receptor (FRalpha) was significantly overexpressed in clinically nonfunctional (NF) adenomas but not in functional adenomas (adrenocorticorticotropic hormone, growth hormone, and prolactin). FRalpha is a high affinity folate transporter that is overexpressed by other tumors and could provide a growth advantage to cells that express it. Analysis of FRalpha expression by Western blotting confirmed that FRalpha protein was specifically overexpressed in NF tumors. The FRalpha was capable of binding folates from measurements of [(3)H] folic acid binding, indicating that the overexpressed receptor was properly folded and may mediate vitamin uptake. Comparison of protein and specific [(3)H] folic acid binding levels in subtypes of NF adenomas suggested that the immunohistochemically negative adenomas produced more properly folded FRalpha than adenomas that stained positively for anterior pituitary hormones. Finally, immunohistochemistry demonstrated that FRalpha was specifically expressed in NF adenoma cells. These results demonstrate that overexpression of FRalpha mRNA by NF pituitary adenomas results in production of properly folded FRalpha protein, may mediate vitamin transport, and could potentially facilitate the growth of these tumors.