We examined the metabolic responses of the hypoxia tolerant killifish (Fundulus heteroclitus) to 15 hr of severe hypoxia and recovery with emphasis on muscle substrate usage and the regulation of the mitochondrial protein, pyruvate dehydrogenase (PDH), which controls carbohydrate oxidation. Hypoxia survival involved a transient activation of substrate-level phosphorylation in muscle (decreases in [CrP] and increases in [lactate]) during which time mechanisms to reduce overall ATP consumption were initiated. This metabolic transition did not affect total cellular [ATP], but had an impact on cellular energy status as indicated by large decreases in [ATP]/[ADP_free] and [ATP]/[AMP_free] and a significant loss of phosphorylation potential and Gibbs free energy of ATP hydrolysis (fG'). The activity of PDH was rapidly (within 3 hr) decreased by ~50% upon hypoxia exposure and remained depressed relative to normoxic samples throughout. Inactivation of PDH was primarily mediated via post-translational modification following the accumulation of acetyl-CoA, and subsequent activation of pyruvate dehydrogenase kinase (PDK). Estimated changes in cytoplasmic and mitochondrial [NAD+]/[NADH] did not parallel one another, suggesting the mitochondrial NADH shuttles do not function during hypoxia exposure. Large increases in the expression of PDK (PDK isoform 2) were consistent with decreased PDH activity; however, these changes in mRNA were not associated with changes in total PDK-2 protein content assessed using mammalian antibodies. No other changes in the expression of other known hypoxia responsive genes (e.g. lactate dehydrogenase-A or -B) were observed in either muscle or liver.