The molecular analysis of pituitary tumours has received a great deal of attention, although the majority of studies have concentrated on the genome and the transcriptome. We aimed to study the proteome of human pituitary adenomas. A protein array using 1005 monoclonal antibodies was used to study GH-, corticotrophin- and prolactin-secreting as well as non-functioning pituitary adenomas (NFPAs). Individual protein expression levels in the tumours were compared with the expression profile of normal pituitary tissue. Out of 316 proteins that were detected in the pituitary tissue samples, 116 proteins had not previously been described in human pituitary tissue. Four prominent differentially expressed proteins with potential importance to tumorigenesis were chosen for validation by immunohistochemistry and western blotting. In the protein array analysis heat shock protein 110 (HSP110), a chaperone associated with protein folding, and B2 bradykinin receptor, a potential regulator of prolactin secretion, were significantly overexpressed in all adenoma subtypes, while C-terminal Src kinase (CSK), an inhibitor of proto-oncogenic enzymes, and annexin II, a calcium-dependent binding protein, were significantly underexpressed in all adenoma subtypes. The immunohistochemical analysis confirmed the overexpression of HSP110 and B2 bradykinin receptor and underexpression of CSK and annexin II in pituitary adenoma cells when compared with their corresponding normal pituitary cells. Western blotting only partially confirmed the proteomics data: HSP110 was significantly overexpressed in prolactinomas and NFPAs, the B2 bradykinin receptor was significantly overexpressed in prolactinomas, annexin II was significantly underexpressed in somatotrophinomas, while CSK did not show significant underexpression in any tumour. Protein expression analysis of pituitary samples disclosed both novel proteins and putative protein candidates for pituitary tumorigenesis, though validation using conventional techniques are necessary to confirm the protein array data.