In a series of three papers, we demonstrate and validate an approach for concurrent absolute quantification in situ of blood flow and energy metabolism with a modification of the NMR methodfor absolute concentration determination put forth by Thulborn and Ackerman [J. Magn. Reson. 55, 357 (1983)] and later expanded upon by Tofts and Wray. In this first paper of the series, we briefly review the theoretical basis for the concentration measurement and present, for the first time, a successful paired validation of metabolite quantification via 31P surface‐coil NMR through corroborative in vitro enzymatic assays. The paired radiolabeled microsphere validation of blood flow measurement via 2H surface‐coil NMR employing D2O as a freely diffusible tracer and the concurrent determination of blood flow and energy metabolism in a septic rat model are presented in the accompanying second and third paper to completethe series. In this article aclassical RF tank circuit is employed to describe the effect of conductive sample loading on the NMR receiver by considering its apparent series resistance. It is shown in aneasily visualized generalizable manner that the effect of sample loading on the observed NMR signal intensity can be accounted for quantitatively by monitoring changes in 90° pulse width at constant power at a fixed reference point, i.e., SSample= Sphantom(PW90pphantom/PW90sample). In a series of paired experiments the absolute concentrations of high energy phosphates obtained from resting rat leg muscle (n = 4) in situ (NMR) and in vitro (enzymatic) were determined as follows: [PCr]NMR = 17.2 ± 0.8 SD, [PCT]enzymatic, = 17.3 ± 2 SD, [ATP]NMR = 5.1 ± 0.8 SD, [ATP]enzymatic, = 5.0 ± 0.2 SD mmol/kg tissue wet wt. Results of these two independent methods of concentration determination were not statistically different (P = 0.94 and P = 0.74 respectively) and serve to rigorously validate the Thulborn approach for absolute quantification of phosphorous metabolites in situ via NMR. Furthermore, these results strongly suggest that ATP and PCr in resting rat leg muscle under normal physiologic conditions are 100% NMR visible. The free cytosolic [ADP]NMR was estimated from the creatine kinase reaction equilibrium expression to be 0.022 ± 0.003 SD mmol/kg tissue wet wt.