Resistance exercise acutely elevates endogenous levels of circulating testosterone (Ratamess et al. 2005), eliciting an anabolic effect, and leading to increases in muscle strength and fibre hypertrophy. The molecular mechanisms by which testosterone (T) contributes to the anabolic process during hypertrophy in skeletal muscle remains poorly understood. Various molecular pathways to promote muscle growth and hypertrophy have been suggested, including directly via the androgen receptor (AR) or indirectly through insulin-Like Growth Factor-I (IGF-I)). However results still remain inconclusive. Therefore we investigated the effect of testosterone on molecular markers of hypertrophy. A confluent monolayer culture of mouse C2C12 skeletal muscle cells was exposed to standard low serum conditions. Treatment consisted of a vehicle control, testosterone (50nM and 500nM) or IGF-I (10ng/ml) for 3 days’ (early) and 6 days’ (late) muscle differentiation. For both time points, reverse transcriptase-polymerase chain reaction (RT-PCR) analysis was performed for AR, IGF-I and myogenin (terminal marker of differentiation) messenger RNA (mRNA) and immunocyctochemistry to determine myotube width (μm), fusion index (%) etc. The experiment was performed in triplicate, with 3 separate repeats (n=3). Values are means ± S.D, compared by ANOVA. After 3 days, myogenin mRNA expression significantly increased with exogenous T treatment (50nM T 1±0.3; 500nM T 1.1±0.4 (p<0.01 respectively)). No further changes occurred in myogenin mRNA levels after 6 days exposure in any of the treatments. As for AR mRNA expression, there were no significant changes between testosterone doses at either time points, where as exogenous IGF-I significantly increased AR mRNA expression after 3 days exposure (1.55±0.82, p<0.05). Finally, following 6 days exposure, both doses of testosterone significantly increased myotube width (50nM T 21.8±5.9μm; 500nM T 21.1±5.5μm (p<0.05 respectively)) compared to control (18.08±3.6μm) and IGF-I (17.8±4.3μm) treatment. The present study supports testosterone’s role in myogenic differentiation. Interestingly, the 50nM dose exerted a greater effect in this cellular process. An observation potentially explained by testosterone-androgen receptor interactions and the reported (Altuwaijri et al. 2004) low levels of AR in C2C12 cells. This data also supports in vivo results demonstrating increased fibre diameter during testosterone supplementation (Hartgens et al, 2002). Furthermore varying levels of AR have been observed between muscle groups (Kadi et al. 2000), optimising training regimes for such muscle groups may be important to maximise testosterones hypertrophic effect on skeletal muscle.