In a recent study we showed that treating isolated intact mouse fast- and slow-twitch skeletal muscle fibres with physiological levels of dihydrotestosterone (DHT), for one hour, leads to an increase in maximum isometric tension (Po) in the fast twitch fibres and a decrease in Po in the slow twitch fibres (Mutungi, 2008). However, in that study the molecular mechanisms underlying these effects were not investigated. Therefore, the primary aim of this study was to investigate the molecular mechanisms underlying the effects of DHT on Po in mouse skeletal muscle fibre bundles. All the experiments were performed at 20°C using small muscle fibre bundles isolated from either the extensor digitorum longus (edl, a mainly fast twitch muscle) or soleus (a predominantly slow twitch muscle) of adult female CD-1 mice 49 ± 4 (n=8; S.E.M) days. The mice were humanely killed and all the experiments conformed to the Animals (Scientific Procedures) Act 1986. The fibre bundles were treated for 1hr with either 630ρg/ml DHT dissolved in absolute ethanol (experimental) or the vehicle only (6.3μL/100ml; controls). In another experiment, the bundles were pre-treated for 30 minutes with inhibitors of either the epidermal growth factor receptor [EGFR]) (100ηM AG1478) or the androgen receptor (AR) (3µM flutamide and 1μM cyproterone acetate) before treatment with DHT plus the inhibitor for 1hr. At the end of each experiment, the bundles were processed for immunoblotting and the changes in the phosphorylation of extracellular signal regulated kinases 1&2 (pERK1&2) and myosin light chains (pMLCs) were probed using antibodies against the phosphorylated proteins. The findings show that treating the fibre bundles with DHT led to a 2-3 fold increase in the phosphorylation of ERK 1&2 in both fibre types and MLC in the fast twitch fibres only. Moreover, this increase was blocked by AG1478 but not by flutamide and cyproterone acetate. From these results we suggest that DHT modulates force production in mammalian skeletal muscles by regulating the phosphorylation of MLCs by ERK1&2.