Several studies have demonstrated the role of Wnt and Notch signaling in the pathogenesis of pituitary adenomas, but data are scarce regarding the role of Hedgehog signaling. In this study we investigated the differential expression of gene targets of the Hedgehog signaling pathway. Formalin-fixed, paraffin-embedded specimens from adult patients who underwent transphenoidal resection and normal human pituitary tissues that were obtained from autopsies were used. Clinical information and data from pre-operative MRI scan (extracellular tumor extension, tumor size, displacement of the optic chiasm) were retrieved from the Hospital's database. We used a customized RT(2) Profiler PCR Array, to investigate the expression of genes related to Notch and Hedgehog signaling pathways (PTCH1, PTCH2, GLI1, GLI3, NOTCH3, JAG1, HES1, and HIP). A total of 52 pituitary adenomas (32 non-functioning adenomas, 15 somatotropinomas and 5 prolactinomas) were used in the final analysis. In non-functioning pituitary adenomas there was a significant decrease (approximately 75%) in expression of all Hedgehog related genes that were tested, while Notch3 and Jagged-1 expression was found significantly increased, compared with normal pituitary tissue controls. In contrast, somatotropinomas demonstrated a significant increase in expression of all Hedgehog related genes and a decrease in the expression of Notch3 and Jagged-1. There was no significant difference in the expression of Hedgehog and Notch related genes between prolactinomas and healthy pituitary tissues. Hedgehog signalling appears to be activated in somatotropinomas but not in non-functioning pituitary adenomas in contrast to the expression pattern of Notch signalling pathway.