The transduction function for ADP stimulation of mitochondrial ATP synthesis in skeletal muscle was reconstructed in vivo and in silico to investigate the magnitude and origin of mitochondrial sensitivity to cytoplasmic ADP concentration changes. Dynamic in vivo measurements of human leg muscle phosphocreatine (PCr) content during metabolic recovery from contractions were performed by (31)P-NMR spectroscopy. The cytoplasmic ADP concentration ([ADP]) and rate of oxidative ATP synthesis (Jp) at each time point were calculated from creatine kinase equilibrium and the derivative of a monoexponential fit to the PCr recovery data, respectively. Reconstructed [ADP]-Jp relations for individual muscles containing more than 100 data points were kinetically characterized by nonlinear curve fitting yielding an apparent kinetic order and ADP affinity of 1.9 +/- 0.2 and 0.022 +/- 0.003 mM, respectively (means +/- SD; n = 6). Next, in silico [ADP]-Jp relations for skeletal muscle were generated using a computational model of muscle oxidative ATP metabolism whereby model parameters corresponding to mitochondrial enzymes were randomly changed by 50-150% to determine control of mitochondrial ADP sensitivity. The multiparametric sensitivity analysis showed that mitochondrial ADP ultrasensitivity is an emergent property of the integrated mitochondrial enzyme network controlled primarily by kinetic properties of the adenine nucleotide translocator.