The aim of this study was the quantification of inorganic phosphate (Pi) and other phosphorus metabolites by P-31 NMR spectroscopy in the mouse heart muscle in situ, beating at around 600 min(-1). Male adult Quacker-bush mice (mean weight 32 +/- 7 g) were anaesthetized, ventilated and placed in a temperature-controlled animal holder. A purpose-built 31P NMR surface coil was positioned against the exposed left ventricular myocardium. Partial signal overlap of Pi with 2,3-DPG from chamber blood was minimized using a DEPTH pulse sequence (180 degrees -90 degrees -180 degrees -180 degrees -acq.). Quantification of phosphorus metabolites was performed using an external standard positioned directly above the surface coil. We report for the mouse myocardium in situ an intracellular free [p(i)] of <0.4 mM, pH of 7.32 +/- 0.1, free [Mg2+] of 0.41 +/- 0.1 mM, free [ADP] of 13 +/- 1.5 <mu>M, [ATP] of 5 +/- 0.5 mM and [PCr] of 14 +/- 1,5 mM. The phosphorylation ratio (ATP/ADP P-i) was 1005 +/- 200 mM(-1) for a PCr/ATP ratio of 2.7 +/- 0.3, It was concluded that the detection of free [P-i] in the mouse myocardium in situ can be greatly enhanced using a DEPTH pulse sequence. Quantification of compounds using an external standard positioned directly above the surface coil gave comparable results to estimations using internal ATP that was quantified enzymatically. The close agreement between the external and internal methods indicates that ATP is 100% NMR visible in the mouse heart in situ. Copyright (C) 2000 John Wiley & Sons, Ltd.