The etiology of sporadic pituitary tumors is currently unknown. The Wnt pathways have been implicated in the pathogenesis of a variety of human tumors but the role of these pathways in pituitary tumors is unclear. Microarray analysis using the Affymetrix HG U133 plus 2.0 GeneChips identified four secreted frizzled-related protein (sFRP) family members of Wnt pathway inhibitors that were differentially expressed in both nonfunctioning and clinically functioning pituitary tumors (n=20) compared with normal pituitary controls (n=3). Reduced tumor expression of Wnt inhibitory factor-1 (WIF1), sFRP2 and sFRP4 mRNA was confirmed by real-time quantitative RT-PCR (p <0.001, p=0.002 and 0.013 respectively) in all pituitary subtypes. Hypermethylation of the WIF1 promoter was present in 88% of the pituitary tumors (n=41). Seventy-six percent of pituitary tumors demonstrated absent or weak cytoplasmic WIF1 staining by immunohistochemistry (n=41), although preserved staining was seen in some functioning tumors, with strong staining in 92% of normal pituitary controls (n= 13). The Wnt pathway target gene, cyclin D1 was found to be up-regulated specifically in the nonfunctioning pituitary tumors compared with controls at both mRNA and protein level, supportive of activation of the Wnt-beta-catenin pathway. Nuclear accumulation of beta-catenin, however, was not observed in any pituitary tumors (n=70). By transfecting GH3 cells with WIF1, decreased cell proliferation and colony formation was observed compared with empty vector controls. In conclusion, our data suggests that WIF1 may be a tumor suppressor, specifically in non-functioning pituitary tumors, and that the Wnt pathways are important in pituitary tumorigenesis.