The hypophysial pars tuberalis (PT), an important interface between neuroendocrine brain centers (hypothalamus, pineal organ) and the pars distalis (PD) of the hypophysis, plays a central role in regulating seasonal reproduction and prolactin release. However, the signaling molecules that transmit photoperiodic information from the PT to the PD and control prolactin release (the so-called "tuberalins") have not yet been identified, despite an intense search for more than three decades. Here, we demonstrate an endocannabinoid system in the PT of the Syrian hamster, a photoperiodic species. By means of in situ hybrization, the PT was found to express N-acylphosphatidylethanolamine-specific phospholipase D (NAPE-PLD), fatty acid amide hydrolase (FAAH), sn-1-selective diacylglycerol lipases (DAGLalpha and DAGLbeta), and monoacylglycerol lipase (MAGL), enzymes involved in endocannabinoid synthesis and degradation. The expression of NAPE-PLD, FAAH, and DAGLalpha was confirmed by immunohistochemistry. Expression and protein levels of DAGLs controlling the synthesis of 2-arachidonoyl glycerol (2-AG), a major endocannabinoid, were upregulated in the PT of Syrian hamsters kept under long-day conditions. Consequently, 2-AG levels were increased in the PT of these hamsters. A primary target of 2-AG, the cannabinoid receptor 1 (CB1), was expressed in the PD. Double-immunolabeling revealed that most of the CB1-immunoreactive cells in the PD were folliculostellate cells that were also immunoreactive for S-100 protein. Thus, the PT comprises an endocannabinoid system, and 2-AG may act as a photoperiodic messenger from the PT to the PD for the regulation of hypophysial hormonal secretion.