Oxygen (O2) is an essential component in maintaining physiological homeostasis within mammalian cells and survival is classically viewed as being dependent upon the counterbalance of O2 supply and O2 demand (Arthur et al., 2000). Oxygen deprivation (hypoxia), such as that experienced at altitude, provides an important challenge to the organism which may severely compromise body metabolism, promoting reversible or irreversible loss of tissue and cell homeostasis leading to organic and functional decay (Magalhães & Ascensão, 2008). Loss in skeletal muscle mass in chronic hypoxia has been demonstrated during in vivo studies by a reduction of muscle (10-15%) and cross sectional area, including a decrease in muscle fibre size (20-25%) above 3000 m (Green et al., 1989; Hoppeler et al., 1990). In vitro studies support these findings demonstrating that chronic hypoxia (> 24 hours) leads to a decrease in cell proliferation and differentiation (Chakravarthy et al., 2001). However, the acute response is substantially understudied and could be important in understanding the effects of hypoxia on skeletal muscle during brief exposures such as those observed within intermittent hypoxic training programmes. The purpose of this work was to examine the role of acute hypoxia (24 h) on C2C12 proliferation and relevant gene expression in 2D culture. 15 x 103 C2C12 myoblasts were seeded into six well plates. The cells were maintained in DMEM with 20% FCS. C2C12 myoblasts were either exposed to 21% or 5% O2 (Figure 1). At 24, 48, 72 and 96 hours RNA was extracted using the Trizol® method and mRNA expression of myogenic regulatory factors, myoD, myf5 and myogenin were detected using the 2-ΔΔCT method. Cell counts and cell viability were also quantified. Results were analysed using a 2 x 2 ANOVA. MyoD, myf5 and myogenin relative mRNA expression was not significantly different (P > 0.05) between the two conditions. Cell counts and cell viability also showed no change in hypoxia compared with normoxia. The data demonstrate that C2C12 myoblasts can be cultured in hypoxia for 24 hours with no significant effect on proliferation. Thus, exposing individuals to 24 hours of hypoxia which results in an intramyocellular O2 pressure of 5% appears safe for use within hypoxic research studies.