Citrate synthase (CS) is a key mitochondrial enzyme in the tricarboxylic acid cycle (TCA). TCA provides NADH and FADH for the ETC to generate ATP through oxidative phosphorylation in muscle cells. The aim of this PhD project is to investigate the role of CS in skeletal muscle metabolism. The aim of the first study was to investigate the effects of a high fat diet (HFD, 45 % kcal fat) for 12 weeks on CS activity in the heart and gastrocnemius muscle of C57BL/6J (B6) mice and congenic (B6.A) characterised by 39% reduced CS activity. Spectrophotometric analysis of CS activity in the heart and gastrocnemius muscle revealed that HFD led to an increase in CS activity in gastrocnemius muscle but a decrease in the heart in both strains of mice. The aim of the second study was to investigate the effects of low CS activity on substrate metabolism in primary muscle cells established from B6 and B6.A mice. Primary muscle cells from both strains were incubated in radiolabelled glucose or palmitate to assess their oxidation in the mitochondria. The reduction of CS activity in B6.A muscle cells did not affect glucose and palmitate oxidation. The aim of the third study was to investigate the effects of D- and L-serine on CS activity in B6 muscle homogenates, primary muscle cells and purified CS from porcine heart. The muscle samples were incubated in D - or L-serine at 0.1 mM or 5 mM concentration and CS activity levels were assessed by spectrophotometer. D- or L-serine did not have any effect on CS activity in muscle samples. The aim of the fourth study was to investigate the effects of low CS activity on substrate metabolism in C2C12 muscle cells. Lentiviral transduction of C2C12 muscle cells with shRNA resulted in a reduction of CS activity and the metabolic pathways were assessed using XF24 Analyser, western blotting, Immunofluorescence and qRTPCR. Low CS activity was associated with a reduction in substrate oxidation by the mitochondria, an increase in glycolysis and ceramide accumulation in C2C12 muscle cells. The results highlight the significance of CS activity as a modulator of muscle metabolism.