We investigated the proteome of the anterior pituitary gland (AP) in a species in which the genome has been sequenced. Subcellular fractions of APs from 2-month-old male mice were prepared for protein denaturation, treatment with trypsin and analyses utilizing micro liquid chromatography tandem mass spectrometry and the database search software SEQUEST. In the nuclear, non-nuclear 100,000 g and cytosolic fractions, we identified 49, 36 and 68 different proteins, respectively. A total of 115 distinct proteins were detected. We identified growth hormone, prolactin, pro-opiomelanocortin, the α-subunit for the glycoprotein hormones, and luteinizing hormone-β. Groups of other identified proteins included hormone-processing, secretion granule-associated, non-hormonal endoplasmic reticulum-associated, calcium-binding, protein kinase C-associated, histones, non-histone chromosomal, other RNA-binding, heterogeneous nuclear ribonucleoproteins, splicing factors, helicases, lamins, ribosomal, microtubule-associated, microfilament-associated, adenosine triphosphate- and guanosine triphosphate-associated, tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation, enzymes in glycolysis and the tricarboxylic and urea cycles and the pentose phosphate path, heat-shock, glutathione-associated, peroxidases, ubiquitin-associated, catabolic, protease inhibitors, other, and blood proteins. The 115 proteins reported in this study and the 145 proteins reported in a previous study on the AP of the adult male Golden Syrian hamster are compared and form a foundation for defining the proteome in normal adult male AP.