The aim of this study was to examine two methods of ³¹P NMR quantitation of phosphocreatine (PCr), ATP, and P_i in rat heart and skeletal muscle in vivo. The first method employed an external standard of phenylphosphonic acid (PPA; 10 mM), and the second method used an enzymatic measurement of tissue ATP equated to the area under the ATP peak. With the use of the external standard, the concentrations of ATP, PCr, and P_i in the rat heart were 4.48 ± 0.33, 9.21 ± 0.65, and 2.25 ± 0.16 µmol/g wet wt, respectively. With the use of the internal ATP standard, measured on the same tissue, the contents (means ± SE) were 4.78 ± 0.19, 9.83 ± 0.18, and 2.51 ± 0.33 µmol/g wet wt, respectively (n = 7). In skeletal muscle, ATP, PCr, and P_i were 6.09 ± 0.19, 23.44 ± 0.88, and 1.81 ± 0.18 µmol/g wet wt using the PPA standard and 6.03 ± 0.19, 23.30 ± 1.30, and 1.82 ± 0.19 µmol/g wet wt using the internal ATP standard (n = 6). There was no significant difference for each metabolite as measured by the two methods of quantification in heart or skeletal muscle. The results validate the use of an external reference positioned symmetrically above the coil and imply that each has similar NMR sensitivities (similar signal amplitude per mole of ³¹P between PPA and tissue phosphorus compounds). We conclude that PCr, ATP, and P_i are nearly 100% visible in the normoxic heart and nonworking skeletal muscle given the errors of measurement.