Hexose-6-phosphate dehydrogenase is an important factor in setting the redox status of the endo-/sarcoplasmic reticulum (ER/SR) lumen by generating the NADPH:NADP+ ratio for 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) mediated glucocorticoid (GC) activation. H6PDH knockout mice (H6KO) clearly demonstrate the obligate nature of 11β-HSD1 for H6PDH, and display a vacuolating of type IIb fiber myopathy, elevated glycogen storage and type II to type I fibre type switching. They also display fasting hypoglycaemia, GC insensitivity and reduced skeletal muscle mass. To identify factors interacting with H6PDH and initiating myopathy, we have applied targeted metabolomics technology for quantification of 163 metabolites in quadriceps muscle from 8 weeks old H6KO and control mice (n=10). Of the 163 metabolites, 14 were amino acids of which Arg, Trp, Gly, Gln, and Pro were significantly elevated whereas Orn and xLeu significantly decreased. These changes reflected a diminished Orn:Arg ratio representing greatly impaired arginase activity. Additionally, we have screened the mRNA expression of most differentially regulated genes previously identified in H6KO myopathy-affected muscle by real-time PCR, and we have found a set of significantly deregulated genes involved in amino acids metabolism, with Gls, Gpt, Smox1, and Amd1 expression was decreased and Pycr1 and Aldh18a1 expression increased.Our preliminary data suggest that H6KO impacts amino acid metabolism that could contribute to aspects of the phenotype such as insulin sensitisation, such as elevated arginine and subsequent nitric oxide content, whereas changes in protein synthesis and amino acid accumulation might lead to a reduction in muscle mass and influence ER stress. These data are being analysed further using informatic approaches and may offer insight into homeostatic responses on amino acid metabolism during metabolic stress in skeletal muscle.