The actin-binding protein α-actinin-3 is one of the two isoforms of α-actinin that are found in the Z-discs of skeletal muscle, and is specifically expressed in fast glycolytic muscle fibres. Homozygosity for a common polymorphism in the ACTN3 gene results in complete deficiency of α-actinin-3 in about 1 billion people worldwide. Although α-actinin-3 deficiency does not cause disease, recent studies suggest that the absence of α-actinin-3 is detrimental to sprint and power performance in elite athletes (Yang et al., 2003). To determine the effect of α-actinin-3 deficiency on the contractile properties of skeletal muscle, we studied isolated extensor digitorum longus muscles (EDL) from a specially developed α-actinin-3 knockout mouse. Animals aged 8 to 10 weeks were sacrificed with an overdose of halothane (ethics approval UNSW). The EDL muscle was dissected from the hindlimb and tied by its tendons to a force transducer at one end and a tissue puller at the other. It was placed in a bath continuously superfused with oxygenated Krebs solution. The muscle was stimulated by two parallel platinum electrodes. At the start of the experiment, the muscle was set to the optimum length L₀ that produced maximum twitch force. All experiments were conducted at room temperature (~ 22°C to 24°C). Each set of experiments was carried out on 10 wild-type and 10 knockout muscles (n=10). All tests were two-tailed t-tests with a significance level of 5%. Data are presented as Mean ±S.E.M. α-Actinin-3-deficient muscles showed similar levels of damage to wild-type muscles following eccentric contractions of 20% strain, 1.6 ± 2.0% in wild-types and 2.6 ± 1.5% in knockouts, suggesting that the presence or absence of α-actinin-3 does not influence the mechanical stability of the sarcomere. α-Actinin-3 deficiency does not result in a loss of fast glycolytic fibres (expressing myosin 2B). However, α-actinin-3-deficient muscles were 9% lighter than α-actinin-3-positive muscles, with a corresponding 9% reduction in cross-sectional area. Knockouts displayed longer twitch half-relaxation times; the half-relaxation time of 15.7 ± 0.6 ms in knockouts was 2.6 ms longer than the half-relaxation time of 13.2 ± 0.6 ms in wild-types (p = 0.008). α-actinin-3-deficient muscles showed significantly better recovery from fatigue; 30 minutes following the fatigue protocol knockouts recovered to 86.1 ± 1.1% of their original force, but wild-types recovered to only 78.4 ± 1.9% of original (p = 0.013). In combination, these data suggest that α-actinin-3 deficiency results in fast-twitch, glycolytic fibres developing slower-twitch, more oxidative properties while not affecting the mechanical strength of the fibre. This alteration towards a slow contractile profile of the fast muscle would be detrimental to optimal sprint and power performance but beneficial for endurance activities.