Phospholipids of 16 malignant and 11 non-malignant human colon specimens were analyzed using a chloroform-methanol analytical reagent in conjunction with 31P magnetic resonance spectroscopy (MRS) at 202.4 MHz. Sixteen individual generic phospholipids were identified and quantified for statistical intergroup comparisons. Statistically significant elevations in the relative concentrations of lysophosphatidylcholine and phosphatidylcholine plasmalogen were seen in malignant tissues along with significantly depressed levels of sphingomyelin and phosphatidylethanolamine plasmalogen. The malignant and non-malignant tissue groups were further differentiated by the detection of the minor phospholipids, lysophosphatidylcholine plasmalogen, lysophosphatidylethanolamine plasmalogen, lysophosphatidic acid and phosphatidylglycerol exclusively present in the malignant tissues and by significant changes in computed phospholipid metabolic indices that were dominated by choline containing lipids. The 31P MRS methods used represent an advancement over previous protocols for identifying and quantifying major and minor tissue phospholipids making this the first direct study of membrane phospholipids in human colon tissues using 31P MRS. The phospholipid profiles obtained may provide important information regarding the nature of the malignant cell's membrane system and identify markers which may be used to estimate malignant propensity, aggressiveness of disease and provide prognostic information.