We have studied the effects of glutamate receptor agonists on the cytosolic Ca2+ concentration ([Ca/+]i) of single rat anterior pituitary (AP) cells. Ionotropic (NMDA and kainate/AMPA) and, to a smaller extent, metabotropic glutamate receptors were both present in all the five AP cell types, defined by the hormone they store. Cells within all the types responded also to thyrotropin-releasing hormone (TRH). Alternative typing by the response to four well-established hypothalamic releasing hormones (HRHs), GHRH, GnRH, CRH, and TRH, was performed. One-third of the cells were not sensitive to any HRH, another third were sensitive to only one HRH, and the last third were sensitive to more than one HRH, frequently to all four. Only the cells responding to TRH showed functional glutamate receptors. Superimposed to the above association, the strongest responses to glutamate were found in the cells responsive to multiple HRHs. These results suggest that glutamate may act, by a nonsynaptic mechanism, as a new releasing factor for one or, like TRH, several AP hormones. Coexpression of glutamate and TRH receptors in the subpopulation of cells responsive to multiple HRHs might have a functional meaning, perhaps related to phenotypic plasticity and long-term regulation of hormone secretion by the anterior pituitary.