The reduction of extracellular oxidants by intracellular electrons is known as trans-plasma membrane electron transport (tPMET). tPMET is prevalent in most organisms and plays a role in cell growth, iron metabolism, cell signaling, and defense. tPMET has also been shown to contribute to the development of many diseases. The goal of this project was to elucidate the mechanism of tPMET in C2C12 myotubes and determine the role this process plays in skeletal muscle. To illuminate the mechanism, I utilized cell-impermeable extracellular electron acceptor, water-soluble tetrazolium salt-1 (WST-1). Superoxide dismutase in the incubation medium or exposure to NADPH oxidase (NOX) isoform 1/4 inhibitor suppressed WST-1 reduction by 70%. This suggests that the primary mechanism of tPMET in C2C12 myotubes is due to the production of superoxide through NOX enzymes. There was a positive correlation between glucose concentration and WST-1 reduction suggesting tPMET is a glucose-sensing process in C2C12 myotubes. WST-1 reduction was also decreased by an inhibitor of the pentose phosphate pathway, dehydroepiandrosterone. In contrast, glycolytic inhibitors, 3PO and sodium fluoride, did not affect WST-1 reduction. Thus, it appears that glucose uptake and processing in the pentose phosphate pathway drives NOX-dependent tPMET. In conclusion, tPMET in C2C12 myotubes is a glucose-sensing process that utilizes the pentose phosphate pathway to produce NADPH which is used by NOX enzymes to generate superoxide. The next aim of this study was to determine a functional role of superoxide once it is produced from tPMET. Reactive oxygen species have been shown to activate various signaling pathways. Western blot analysis demonstrates p70S6k phosphorylation is glucose-dependent in C2C12 myotubes, while the phosphorylation of AKT and MAPK did not differ in the presence or absence of glucose. However, phosphorylation of p70S6k was dependent upon NOX enzymes. Taken together, the data suggest that muscle cells have a novel glucose-sensing mechanism dependent on NADPH production and NOX activity, culminating in increased p70S6k phosphorylation.