A method for the dynamic quantification of glycerophosphorylcholine (GPC) levels in intact tissue by 31P nuclear magnetic resonance spectroscopy was developed and verified by a spectrophotometric technique. Intact tissue nuclear magnetic resonance areas were quantified utilizing an external standard and were corrected for magnetization saturation. Interactive computerized spectral fitting through Lorentzian lineshape analysis and subsequent integration with normalization to the external standard was utilized for the absolute quantification of GPC concentration. Hemodynamically and metabolically uncompromised Langendorff perfused rabbit hearts contained 1.70 +/- 0.23 mumol GPC/g wet wt. This value was not statistically significantly different from the value of 1.45 +/- 0.23 mumol GPC/g wet wt determined by an analytical technique employing glycerophosphoryl-[Me-3H]choline as an internal standard with spectrophotometric quantification. Both methods were accurate with a standard error of 11 and 10%, respectively. The recovery of internal standards utilizing the spectrophotometric technique was 95 +/- 8%. The application of these methods should facilitate the quantification of changes in tissue levels of glycerophosphorylcholine noted in several disease states.