The molecular pathways that promote the proliferation and maintenance of pituitary somatotrophs and other cell types of the anterior pituitary gland are not well understood at present. However, such knowledge is likely to lead to the development of novel drugs useful for the treatment of various human growth disorders. Although muscarinic cholinergic pathways have been implicated in regulating somatotroph function, the physiological relevance of this effect and the localization and nature of the receptor subtypes involved in this activity remain unclear. We report the surprising observation that mutant mice that selectively lack the M3 muscarinic acetylcholine receptor subtype in the brain (neurons and glial cells; Br-M3-KO mice) showed a dwarf phenotype associated with a pronounced hypoplasia of the anterior pituitary gland and a marked decrease in pituitary and serum growth hormone (GH) and prolactin. anterior pituitary gland ͉ dwarfism ͉ knockout mice V arious neurotransmitter systems have been implicated in regulating somatotroph function and growth hormone (GH) secretion, either by acting on the anterior pituitary gland directly or by modulating the release of GH-releasing hormone (GHRH) or somatostatin from the hypothalamus (for a comprehensive review, see ref. 1). However, the physiological relevance of the individual pathways and the identity and localization of the receptor subtypes involved in mediating these effects are not well understood in most cases.Pharmacological studies suggest that central muscarinic cholinergic pathways play a role in stimulating GH release in experimental animals and humans (1-9). The identification of the molecular pathways and the specific muscarinic ACh receptor (mAChR) subtypes involved in mediating these responses should be of considerable potential therapeutic interest. However, work in this area has been complicated by the existence of 5 molecularly distinct mAChR subtypes (M 1 -M 5 ) that are difficult to distinguish by classical pharmacological tools (10, 11). Moreover, virtually all brain regions express multiple mAChRs in a complex, overlapping pattern (12-15).The M 3 mAChR subtype is expressed at relatively high levels in the hypothalamus but is also found in many other brain regions (16-18). At present, little is known about the physiological relevance of these neuronal M 3 mAChRs. To shed light on this issue, we used Cre/loxP technology to generate a set of mutant mice, referred to as Br-M3-KO mice, that selectively lacked the M 3 mAChR subtype in the brain (neurons and glial cells). Strikingly, Br-M3-KO mice showed a dwarf phenotype, associated with a dramatic reduction in the size of the anterior pituitary gland, a marked decrease in pituitary GH and prolactin levels, and significantly reduced serum GH, insulin-like growth factor-1 (IGF-1), and prolactin levels. Remarkably, treatment of Br-M3-KO mice with CJC-1295, a synthetic GHRH analog (19-21), restored normal pituitary size, serum GH and IGF-1 levels, and longitudinal growth. These data reveal an unexpect...