Hormone secretion by GH3 pituitary cells is regulated by oscillations of the cytosolic Ca2+ concentration ([Ca2+]i), which are driven by electrical activity and modulated by hypothalamic releasing factors. We find that micromolar concentrations of L-glutamate and other acidic amino acids, but not selective excitatory amino acid receptor agonists, increase [Ca2+]i in GH3 cells. Activation by glutamate is blocked by dihydropyridines or removal of extracellular Ca2+ or Na+, but not by tetrodotoxin or excitatory amino acid receptor antagonists. Glutamate also accelerated the entry of Mn2+ used as a Ca2+ surrogate for Ca2+ channels. L-Glutamate and other acidic amino acids were taken up into GH3 cells by an Na(+)-dependent high-affinity transporter. The half-maximal effect of glutamate on [Ca2+]i was reached at concentrations similar to the Km for the glutamate transporter. Moreover, only those amino acids taken up through this transporter were able to increase [Ca2+]i. We propose that electrogenic entry of Na(+)-glutamate depolarizes the plasma membrane, thus causing an increase of action potentials firing and Ca2+ entry through voltage-gated channels. Our results suggest that glutamate may cooperate to the modulation of pituitary hormone secretion by an unconventional mechanism involving a high-affinity glutamate transporter rather than excitatory amino acid receptors.