AbstractThe aims of this study were two‐fold: to characterize thein vivo31P NMR spectrum of human breast tumours by identifying the metabolites contributing to each peak; and to demonstrate changes in the detectability of the phosphodiester (PDE) peak at varying field strengths. This was achieved by obtaining31P spectra from 23 patientsin vivoat 1.5 T and also of 11 perchloric acid (PCA) extracts and 3 chloroform‐methanol (CM) extracts of tumour biopsy samples at 9.4 T. In spectra acquiredin vivothe percentage areas for each peak were: phosphomenoester (PME), 15%; Pi, 11%; PDE 35%; phosphocreatine, 5%; γ‐NTP, 11%; α‐NTP, 11% and β‐NTP, 12% of the total spectral area. PCA extracts showed the PME peak to be>70% phosphorylethanolamine whilst the PDE peak included almost equal proportions of glycerophosphorylethanolamine and glycerophosphorylcholine. CM extracts of tumours revealed additional metabolites in the PDE region suggesting that the large PDE peak observedin vivocould arise mainly from phospholipids. Spectra of human breast tumour xenografts examinedin vivoat both 1.9 T and 9.4 T confirmed that the presence of a large PDE peakin vivowas a function of field strength. Further experiments with microsomal membranes from rat mammary tumours at 1.5 and 9.4 T demonstrated that phospholipids are more clearly visible at the lower field strength due to a substantial decrease