Background: Controversy persists concerning the mechanisms and role of general anesthetic inhibition of glutamate release from nerve endings. To determine the generality of this effect and to control for methodologic differences between previous studies, the authors analyzed the presynaptic effects of isoflurane and propofol on glutamate release from nerve terminals isolated from several species and brain regions. Methods: Synaptosomes were prepared from rat, mouse, or guinea pig cerebral cortex and also from rat striatum and hippocampus. Release of endogenous glutamate evoked by depolarization with 20 μM veratridine (which opens voltage-dependent Na+ channels by preventing inactivation) or by 30 mM KCl (which activates voltage-gated Ca2+ channels by membrane depolarization) was monitored using an on-line enzyme-linked fluorometric assay. Results: Glutamate release evoked by depolarization with increased extracellular KCI was not significantly inhibited by isoflurane up to 0.7 mM (∼2 minimum alveolar concentration; drug concentration for half-maximal inhibition > 1.5 mM) or propofol up to 40 μM in synaptosomes prepared from rat, mouse, or guinea pig cerebral cortex, rat hippocampus, or rat striatum. Lower concentrations of isoflurane or propofol significantly inhibited veratridine-evoked glutamate release in all three species (isoflurane IC50 = 0.41-0.50 mM; propofol IC50 = 11-18 μM) and rat brain regions. Inhibition of veratridine-evoked release was insensitive to the γ-aminobutyric acid receptor type A antagonist bicuculline (100 μM) in rat cortical synaptosomes. Conclusions: Isoflurane and propofol inhibited Na+ channel-mediated glutamate release evoked by veratridine with greater potency than release evoked by increased KCI in synaptosomes prepared from three mammalian species and three rat brain regions. These findings are consistent with a greater sensitivity to anesthetics of presynaptic Na+ channels than of Ca2+ channels coupled to glutamate release. This widespread presynaptic action of general anesthetics is not mediated by potentiation of γ-aminobutyric acid type A receptors, though additional mechanisms may be involved.