The use of 31P MRS in clinical cancer research has been hampered by both poor anatomic localization of spectra and poor resolution of overlapping signals. We found that accurate localization using 3D chemical shift imaging and improved resolution using 1H-decoupling and nuclear Overhauser-enhancement (NOE) increased signal-to-noise and permitted resolution of separate components within phosphomonoester (PME) and phosphodiester (PDE) regions. Fifty-three cancers of different types (lymphoma, sarcoma, adenocarcinoma) had the following common features: (1) phosphoethanolamine the dominant PME; (2) glycerophosphoethanolamine and -choline rarely detected; (3) a broad PDE signal probably from membrane phospholipids; and(4) prominent nucleoside triphosphates. 1H-decoupling with NOE-enhancement permitted us to obtain new information about in vivo metabolism in human cancers; generate new hypotheses and help guide development of experimental models appropriate to test them; and provide a firm basis with which to examine clinical uses of 31P MRS.