Although glutamate is the predominant excitatory amino acid in the vertebrate central nervous system (CNS) where it affects a variety of physiological processes and pathophysiological states, the role that glutamate receptors may play outside the CNS has not been clearly established. In the present study, the effects of N-methyl-D-aspartate (NMDA), α-amino-2,3-dihydro-5-methyl-3-oxo-4-isoxazolepropanoic acid (AMPA) kainate, and metabotropic glutamate receptor agonists and antagonists were investigated on neuroendocrine melanotropes of the rat pars intermedia using single-cell dual-wavelength microfluorometry and the Ca2+-sensitive probe, fura-2, to monitor changes in [Ca2+]i. Glutamate induced a rapid, concentration-dependent rise in [Ca2+]i with an EC50 of 24 µM that was Mg2+-sensitive and dependent on the presence of extracellular Ca2+. NMDA increased [Ca2+]i in a glycine-dependent manner with an EC50 of 83 µM that was blocked by 1 µM MK-801 and 1 mM Mg2+. The non-NMDA receptor agonists kainate, AMPA, and quisqualate increased [Ca2+]i with an EC50 of 124, 5 and 8 µM, respectively. Responses to kainic acid were blocked by 10 µM CNQX and were shown to be sensitive to Mg2+ and dihydropyridine. AMPA stimulation was the most potent, and glutamate stimulation was the most efficacious at mediating increases in [Ca2+]i. The metabotropic receptor-specific agonist, trans-ACPD, failed to induce a change in [Ca2+]i. The glutamate-induced Ca2+ influx was about half of that elicited by a 50 mM K+-induced membrane depolarization and activation of voltage-sensitive Ca2+ channels. These results demonstrate the presence of glutamate receptors on rat melanotropes and suggest that glutamate receptors in the intermediate lobe of the pituitary may provide the excitatory counterbalance to the well-described secretoinhibiting input via dopamine and γ-aminobutyric acid receptors.