Cocaine and amphetamine regulated transcript (CART) is a highly expressed peptide implicated in the regulation of feeding, reward and reinforcement, and stress-related behaviors. CART has been localized to discrete cell populations in the brain, gut, adrenal gland, and pancreas. In contrast, CART producing cell types in the pituitary gland remain ill defined. In the present study, double-label immunohistochemistry, employing high affinity antiserum we generated against CART (62-102), was used to identify CART producing cells in the pituitary gland. In the anterior pituitary, the majority of CART immunoreactivity (ir) was localized in lactotropes; minor populations of CART immunoreactive cells were identified as somatotropes and corticotropes. In the posterior pituitary, CART-ir extensively colocalized with oxytocin containing fibers; in contrast, only a few vasopressin fibers contained CART-ir. As expected, CART colocalized with oxytocin in magnocellular neurons of the supraoptic nucleus. The effects of bromocryptine, a potent dopamine receptor agonist, were examined to determine if CART mRNA expression and protein release are regulated in a similar fashion to prolactin. Similar to prolactin, CART mRNA expression and protein release were significantly decreased following bromocryptine treatment of dispersed rat anterior pituitary cells in culture. To further explore the putative physiological role of pituitary CART, we compared levels of CART mRNA expression in lactating and non-lactating female rats. CART mRNA levels were significantly increased in the anterior pituitary and supraoptic nucleus of lactating rats. Furthermore, levels of CART in the systemic circulation were significantly elevated at the onset of lactation, peaked at day ten of lactation and returned to baseline values ten days after pups were weaned. The current study describes the cellular localization and regulation of CART expression and protein release from the rat pituitary gland. These findings suggest a putative role for CART in lactation.