Cannabinoids are known to exert effects throughout the endocrine system, including in the anterior pituitary. Whilst cannabinoid receptors (CB1 in particular) are expressed in numerous tissues, expression and function of an endocannabinoid system within specific anterior cell types has not been investigated. We have used well-characterised gonadotroph (LβT2 and αT3-1) and somatolactotroph (GH3) cell lines to establish the expression and functional role for cannabinoid signalling. Initial RT-PCR studies established that LβT2 cells expressed Cnr1, as well as the cannabinoid synthetic enzymes, Faah, Mgll and Napepld. αT3-1 and GH3 cells, and primary mouse pituitary tissue only expressed some of these components. Having established that LβT2 cells expressed a complete endocannabinoid system, functional studies were undertaken. As the CB1 (Cnr1) receptor couples to Gαi in many tissues, the potential inhibitory effect of CB1 signalling was determined in LβT2 cells. Following transient transfection with reporter genes expressing either the human αGSU promoter, the murine Egr-1 promoter, or a Gal4CREB two-hybrid system, cells were stimulated with 0 or 10 μM Forskolin (FSK, adenylyl cyclase activator), in the absence or presence of a range of concentrations of HU210 (a CB1 agonist). HU210 caused an inhibition of the αGSU and Gal4CREB response to FSK, but failed to alter Egr-1 promoter activity. Similar studies in GH3 cells revealed dramatic inhibition in FSK-stimulated αGSU promoter activity in the presence of HU210, suggesting that CB1 couples to Gαi in both LβT2 and GH3 cells. As CB1 receptors can couple to numerous G-protein α-subunits, dynamic confocal imaging of intracellular calcium concentrations ([Ca2+]i) was performed, following Fluo4A/M loading. HU210 caused a rapid increase in [Ca2+]i in LβT2 cells, suggesting potential coupling to Gαq/11. Collectively, these data reveal gonadotrophs and somatolactotroph lineage cells to be putative targets of (endo)cannabinoid signalling in the pituitary, which may reveal potential therapeutic benefits for fertility and growth disorders.