Cocaine- and amphetamine-regulated transcript (CART) was originally isolated from rat brain but CART is also synthesized and stored in the anterior pituitary. The localisation of pituitary CART and factors regulating its synthesis are largely unknown. The regulation of pituitary CART synthesis and release in response to CRF and glucocorticoids was examined in vitro and in vivo. CART-immunoreactivity (CART-IR) was released from anterior pituitary segments. This release was increased fifteen fold in response to CRF. Intraperitoneal administration of CRF to rats significantly increased plasma CART-IR. Furthermore, CART-IR content and plasma CART-IR were significantly increased in adrenalectomised rats and anterior pituitary CART mRNA expression, CART-IR content and plasma CART-IR significantly decreased in corticosterone-treated rats. Plasma CART-IR showed a pattern of diurnal variation similar to that of ACTH and corticosterone and plasma CART-IR was positively correlated with corticosterone. CART-IR was detectable in the medium of the corticotroph cell line, AtT-20. Dual in situ hybridisation for preproCART mRNA expression and immunocytochemistry for ACTH showed localisation of preproCART mRNA to a subpopulation of ACTH immunoreactive cells. These findings demonstrate that pituitary CART expression and release are regulated by CRF and the glucocorticoid environment and pituitary CART is partly localised to corticotrophs.