Rationale: 31P magnetization saturation transfer (MST) experiment is the most widely used method to study ATP metabolism kinetics. However, its lengthy data acquisition time greatly limits the wide biomedical applications in vivo, especially for studies requiring high spatial and temporal resolutions. Objective: We aimed to develop a novel superfast MST method that can accurately quantify ATP production rate constants (kf) through creatine kinase (CK) or ATP synthase (ATPase) with 2 spectra. Methods and Results: The T1nom (T1 nominal) method uses a correction factor to compensate the partially relaxed MST experiments, thus allowing measurement of enzyme kinetics with an arbitrary repetition time and flip angle, which consequently reduces the data acquisition time of a transmurally differentiated CK kf measurement by 91% as compared with the conventional method with spatial localization. The novel T1 method is validated theoretically with numeric simulation, and further verified with in vivo swine hearts, as well as CK and ATPase activities in rat brain at 9.4 Tesla. Importantly, the in vivo data from swine hearts demonstrate, for the first time, that within an observation window of 30 minutes, the inhibition of CK activity by iodoacetamide does not limit left ventricular chamber contractile function. Conclusions: A novel MST method for superfast examination of enzyme kinetics in vivo has been developed and verified theoretically and experimentally. In the in vivo normal heart, redundant multiple supporting systems of myocardial ATP production, transportation, and utilization exist, such that inhibition of one mechanism does not impair the normal left ventricular contractile performance. © 2011 American Heart Association. All rights reserved.