The mammalian pineal gland uses L-glutamate as an intercellular chemical transmitter to regulate negatively melatonin synthesis. To receive glutamate signals, pinealocytes express at least three kinds of glutamate receptors: metabotropic receptor types 3 and 5 and an ionotropic receptor, GluR1. In this study, we examined whether or not the fourth class of ionotropic receptor, delta, which is known for its nondefinitive molecular function and its unique expression pattern in brain, is expressed in pineal gland. RT-PCR analyses with specific probes indicated the expression of mRNA of delta2 but not that of delta1 in pineal gland and cultured pineal cells. Western blotting analysis with polyclonal antibodies specific to the carboxyl-terminal region of the delta2 receptor recognized a single 110-kDa polypeptide of cerebellar membranes and specifically immunostained Purkinje cells. The delta2 antibodies recognized a 110-kDa polypeptide of pineal membranes and specifically immunostained huge glial-like cells with the occasional presence of several long, branching processes in a pineal cell culture. delta2 is not uniformly distributed throughout the cells and is relatively abundant at the periphery of the cell bodies and long processes, where the terminals of synaptophysin-positive processes of pinealocytes, a site for glutamate secretion, are frequently present. The delta2-positive cells constitute a very minor population among total pineal cells (approximately 0.03%). Double immunolabeling with delta2 antibodies and antibodies against marker proteins for pineal interstitial cells clearly distinguishes delta2-positive pineal cells and other known interstitial cells, including glial fibrillary acidic protein- or vimentin-positive glial-like cells. These results indicated that the delta2 glutamate receptor is expressed in a novel subpopulation of pineal glial-like cells in culture and suggest the presence of a glutamate-mediated intercellular signal transduction mechanism between pinealocytes and delta2-expressing cells. The pineal cells may provide a good experimental system for studies on the function of glutamate receptor delta2.