These studies have explored the consequences of activating the prolyl hydroxylase (PHD) oxygen-sensing pathway in twitching neonatal cardiomyocytes. Activation of the PHD pathway was achieved using the prolyl hydroxylase inhibitor (PHI), dimethyloxaloylglycine. PHI treatment of cardiomyocytes caused a decrease in oxygen consumption of 85% and an increase in lactic acid production of 300% in basal conditions. This indicates a ~75% decrease in ATP turnover as the increased ATP generation by glycolysis is inadequate to compensate for the lower respiration. To determine the extent to which decreased ATP turnover underlies the suppressed oxygen consumption, mitochondria were uncoupled with 2, 4-dinitrophenol (DNP). Surprisingly, DNP failed to cause an increase in oxygen consumption by PHI-treated cells, indicating that ETC activity, rather than ATP turnover limits respiration in the PHI-treated cardiomyocytes. Silencing of HIF-1 expression restored the ability of PHI-treated myocytes to increase O₂ consumption when uncoupled; however, basal O₂ uptake rates remained low due to the unabated suppression of cellular ATP consumption. Thus it appears that respiration is actively ‘clamped’ through a HIF-dependent mechanism, while HIF-independent mechanisms are responsible for downregulation of ATP consumption. In addition we find that PHD pathway activation confers mitochondria with the ability to utilize fumarate as a terminal electron acceptor. The source of fumarate for this unusual respiration is derived from the purine nucleotide cycle. In sum, these studies show that the PHD pathway is sufficient to actively ‘clamp’ oxygen consumption and independently suppress ATP consumption. The PHD pathway also equips the mitochondria to utilize fumarate for respiration.