The anterior pituitary gland (AP) secretes six established hormones that collectively control hundreds of biological and behavioral functions. Because of advances in mass spectrometry (MS), protein labeling, and bioinformatics, it is now possible to characterize, compare, and quantify the AP hormones together with large numbers of nonhormonal AP proteins. For example, by using high-performance liquid chromatography in line with tandem MS we characterized 145 proteins in sub-cellular fractions of the AP of young adult male Golden Syrian hamsters and 115 proteins in subcellular fractions of the AP of young adult male mice. These included hormones, proteins involved in hormone synthesis and release, and housekeeping proteins. We also used difference gel electrophoresis in conjunction with MS and peptide mass fingerprinting to quantify the effects of estrogen on the AP-soluble protein fraction in rats. Ovariectomized rats were administered 50 microg of estradiol valerate subcutaneously and studied 48 hrs later, before the onset of the anticipated surges of gonadotropins in blood. Following DeCyder image analysis, we identified by MS and peptide mass fingerprinting 26 protein spots that were upregulated and 19 protein spots that were downregulated. Estrogen increased levels of acidic isoforms of growth hormone and prolactin, several proteins involved in protein synthesis, folding and secretion, and several metabolic enzymes. Most of the downregulated proteins are involved in RNA or DNA interactions. We followed up on the results with RT-PCR and immunohistochemical techniques to demonstrate that one protein identified by MS in hamster AP, fertility protein SP22, is synthesized in the AP and localized primarily in somatotropes and thyrotropes. These experiments demonstrate the efficacy of our proteomics approach to characterize AP proteins and quantify changes in them. The approaches used to study the AP could serve as a model to investigate other heterogeneous organs.