The molecular mechanism of volatile anaesthetic action on presynaptic glutamate release is not clear. An inhibitory effect on voltage-gated calcium channels has been proposed. The present study examines the effect of isoflurane on cytosolic free calcium and synaptic glutamate release from isolated nerve terminals. Synaptosomes from rat cerebral cortex were used. Glutamate was measured with a continuous fluorometric measurement in a spectrophotometer as the fluorescence of NADPH and calcium as the fluorescence of fura-2. Isoflurane reduced the calcium-dependent glutamate release evoked by membrane depolarization with 4-aminopyridine in an inversely dose-dependent manner. The glutamate release was reduced by 56, 43 and 36% in response to isoflurane 0.5, 1.5 and 3.0%, respectively (for all: P < 0.05). Membrane depolarization evoked a rise in cytosolic free calcium of approximately 34%. Addition of isoflurane (0.5, 1.5 and 3.0%) produced no significant change in cytosolic free calcium. These results indicate that the isoflurane-induced reduction in presynaptic glutamate release is caused by other mechanisms than blocking voltage-gated calcium channels. As the release is inversely dose-dependent, two or more mechanisms could be involved.