Background: Absence of the fast-twitch skeletal muscle Z-line protein, α-actinin-3, encoded by the ACTN3 speed gene, is associated with poorer sprinting performance in athletes and a slowing of relaxation in fast-twitch muscles of Actn3 knockout (KO) mice. Around 20% of the world’s population are ACTN3 deficient. Fast-twitch muscles from KO mice display longer twitch half-relaxation times than muscles from wild-type (WT) mice. In mechanically skinned fast fibres the sarcoplasmic reticulum (SR) in fibres from KO mice load calcium more slowly than the SR in fibres from WT mice (Chan et al., 2011). Aim: Our present study investigates the calcium kinetics of fast-twitch fibres from Actn3-KO and WT mice and SERCA pump and parvalbumin protein expression, to see whether any changes in calcium kinetics and associated SR and calcium buffering proteins can account for the previously observed effects of α-actinin-3 deficiency on whole muscle relaxation and SR calcium loading. Methods: Mice were killed with an overdose of halothane, UNSW animal ethics approval 11/140B. Flexor digitorum brevis (FDB) muscles were dissected out and digested in collagense 1a to yield individual fibres. Fibres were plated onto a chamber placed on a Nikon inverted microscope attached to a Cairn spectrophotometer to monitor calcium transients using a photomultiplier tube (PMT). An intracellular microelectrode was used to ionophorese the free acid form of fura-2 or low affinity fura-ff to give a final concentration 5-50 μm. Fibres were electrically stimulated using a bipolar concentric electrode positioned near the neuromuscular junction. In some cases the fibre was immobilized with BTS (4-methyl-N-(phenylmethyl) benzenesulfonamide)to stop contractions Results: In FDB fibres of α-actinin-3-deficient KO mice, the calcium transient in response to a single action potential displayed a lower peak, a slower rate of rise and a faster rate of relaxation than the calcium transients from fibres of WT mice (n=15 for KO, n=19 for WT). Interestingly it was found that KO animals have 50% higher density of SERCA protein while the amount of parvalbumin protein is unchanged. The rate of ICE (calcium release cocktail)-induced SR calcium release was higher in KO. However there does not appear to be a difference in releasable calcium. Calcium-frequency curves showed no significant differences between fibres from KO and WT mice. Conclusions: The faster decay of the calcium transients in fibres of α-actinin-3-deficient KO mice is consistent with data showing an increased density of SERCA pumps in the SR of fibres from KO mice. However, the observations from previous studies that whole fast-twitch muscles from KO mice relax more slowly, and that SR loading is slowed in KO mice, suggest that there may be an increased calcium leakage from the SR of KO mice via the SERCA pump calcium leak pathway.