The skeletal muscle disorder malignant hyperthermia (MH) is associated with increased sensitivity of RYR1 to activation by volatile anaesthetics. Previous work suggests that the inhibitory effect of Mg²⁺ on SR Ca²⁺ release is impaired in MH-susceptible muscle (Laver et al. 1997). In the present study, we have investigated the effects of halothane on Ca²⁺ release from the SR induced by t-tubule depolarisation, in the presence of normal (1 mM) or reduced levels of cytosolic [Mg²⁺]. Rats (200-250 g) were humanely killed. Single extensor digitorium longus (EDL) muscle fibres were mechanically skinned under oil and then superperfused with solutions approximating to the intracellular milieu containing (mM): HDTA, 50; ATP, 8; Na⁺, 37; K⁺,126; phosphocreatine, 10; EGTA, 0.03; Hepes, 90; Ca²⁺, 0.0001; fluo-3, 0.002; pH 7.0, 22°C. Following skinning, the t-tubules reseal and repolarise, allowing the physiological depolarisation-induced Ca²⁺ release process to be initiated by field stimulation. Ca²⁺ release from the SR was measured using line-scan confocal microscopy to detect changes in fluo-3 fluorescence. A train of brief twitch responses was initiated by applying suprathreshold square-wave stimuli (70 V, 2 ms duration) at 2 second intervals, via platinum electrodes running parallel to the fibre. At 1 mM free Mg²⁺, 1 mM halothane induced a significant (17.4 ± 7 %, n=6, mean ± SE, p<0.05) increase in the time taken for the Ca²⁺ transient amplitude to decline by 75%. At 0.4 mM free [Mg²⁺], the effects of halothane were much more pronounced: Ca²⁺ transients failed to decline fully to baseline between stimuli and often exhibited a slow secondary Ca²⁺ release phase, consistent with regenerative Ca²⁺ -induced Ca²⁺ release (CICR). However, in the absence of depolarisation-induced Ca²⁺ release, perfusion with 1 mM halothane failed to induce Ca²⁺ release from the SR at either 1 mM or 0.4 mM free Mg²⁺. These results suggest that in MH, impaired Mg²⁺ inhibition of RYR1 may facilitate the action of halothane on the SR, such that the physiological Ca²⁺ release mechanism fails to terminate. These effects may be of particular importance when susceptibility to MH is combined with (i) hypomagnesemia or (ii) depolarising muscle relaxants.