Carboxypeptidases may play important role(s) in prohormone processing in normal and neoplastic adenohypophyseal cells of the pituitary. We have recently demonstrated carboxypeptidase E (CPE) and carboxypeptidase Z (CPZ) in the majority of adenohypophyseal cells with carboxypeptidase D (CPD) immunoreactivity largely confined to adrenocorticotrophs. This study evaluated the expression patterns of CPE, CPD, and CPZ immunoreactivity in 48 pituitary adenomas. Our immunohistochemistry demonstrated extensive intracytoplasmic immunoreactivity for CPE, CPD, and CPZ in adrenocorticotrophic hormone (ACTH)-producing adrenocorticotroph cells, prolactin-producing lactotroph cells, and growth hormone (GH)-producing somatotroph cell adenomas, all of which require carboxypeptide processing of prohormones to produce active endocrine hormones. In contrast to the restricted expression in the normal adenohypophysis, CPD appeared to be widespread in the majority of adenomas, suggesting that CPD levels are increased in adenomas. In luteinizing hormone/follicle-stimulating hormone (LH/FSH)-producing gonadotroph adenomas, which do not require carboxypeptidases to produce gonadotropins, only CPZ immunostaining was demonstrated. In null-cell adenomas, CPE immunoreactivity was detected in the majority of tumors, but CPD and CPZ were identified only in a minority of cases. CPE in these cells may process other peptides critical for pituitary cell function, such as chromogranin A or B. These findings suggest that CPs participate in the functioning of pituitary adenomas.