Clenbuterol is a β₂-adrenoceptor agonist, structurally similar to adrenaline, used in the treatment of asthma. Although its primary action is bronchodilation, there is growing evidence that it, like other β₂-adrenoceptor agonists, may also have anabolic effects when used for prolonged periods (Lynch & Ryall, 2008). However, the mechanism(s) underlying the anabolic effects of β₂-adrenoceptor agonists is still poorly understood. It is also uncertain whether β₂-adrenoceptor agonists have any direct effects on muscle contraction. Therefore, the primary aim of this study was to investigate the effects of clenbuterol on the contractile properties of isolated intact mouse fast and slow twitch skeletal muscle fibres. All the experiments were performed at 20 ± 0.1°C on small muscle fibre bundles isolated from either the extensor digitorum longus (fast twitch muscle) or soleus (slow twitch muscle) of CD-1 mice 46 ± 1.6 (n=9; S.E.M) days old. The mice were humanely killed and all the experiments conformed to the Animals (Scientific Procedures) Act 1986. The fibre bundles were mounted horizontally between a force transducer and a servomotor and were continuously perfused with mammalian Ringer’s solution. Twitch and tetanic contractions were then recorded in Ringer’s solution with or without any added clenebuterol. In another experiment, the fibre bundles were treated for 1hr with the standard Ringer’s solution or the standard Ringer’s solution with clenbuterol. Proteins isolated from these bundles were then immunoblotted for the levels of phosphorylated AKT, extracellular signal regulated kinases 1&2 (ERK1&2) and phospholamban. At all concentrations investigated (100ηM – 250μM), clenbuterol decreased twitch and tetanic contractions in both fibre types. Used at concentrations <150μM, its effects were completely reversible. However, above this concentration they were not. For example, 50μM clenbuterol reversibly decreased twitch tension to 72 ± 4% and tetanic tension to 23 ± 8% (n=4; S.E.M) of controls in slow twitch fibres. The corresponding values in the fast twitch fibres were 81± 6 and 55 ± 4% (n=5; S.E.M), respectively, of controls. In addition to its effects on force, clenbuterol also increased the phosphorylation of ERK 1&2 and the dephosphorylation of AKT in both fibre types. In contrast, it increased the phosporylation of phospholamban in slow twitch fibres but decreased it in fast twitch fibres. From these results we suggest that clenbuterol decreases force production in mammalian fast- and slow-twitch skeletal muscles by regulating the phosphorylation of phospholamban.