Slices of the molecular layer of the dentate gyrus of rat hippocampus were used to study the regulation of glutamate synthesis and release. Net glutamate synthesis increased dramatically during conditions which stimulated the release of glutamate. The rate of glutamine incorporation into glutamate released into the medium was increased almost immediately upon stimulation with a 56 mM KCl, 3 mM CaCl2 medium. Synthesis appeared to be regulated both by glutamine uptake and the activity og glutaminase. Glutamine uptake was stimulated in the presence of 56 mM KCl and 3 mM CaCl2. The increased glutamine uptake was not due to a decrease in efflux, was unrelated to tissue glutamate levels, and could be dissociated from the rate of glutamate biosynthesis. The presence of Ca2+ ions and depolarization seemed necessary. Glutaminase activity was regulated by end product inhibition: increased levels of tissue glutamate resulted in a decrease in glutamate synthesis. Glutamine in the presence of 56 mM KCl increased the rate of glucose incorporation into glutamate over that seen without glutamine.