End-products of glycolysis as well as phospholipid precursors and catabolites have been suggested as metabolic indicators of tumor progression. To test the hypothesis that increased levels of such indicators can distinguish metastatic phenotypes, we determined a limited cellular 1H-NMR metabolic profile of subpopulations of murine mammary 4T1 cells that differ in their metastatic potential. Subpopulations with differing metastatic phenotypes were identified by sorting for the expression of the cell surface adhesion oligosaccharide sialylated Lewis x (sLeX). The sLeX-negative subpopulation metastasizes to the lung of syngeneic mice more rapidly than the sLeX-positive subpopulations. The metabolic profile of the sLeX-negative subpopulation indicated higher levels of lactate and total choline metabolites than the sLeX-positive subpopulation, suggesting that altered metabolism is a critical component of the malignant phenotype. Analysis of shed cellular material from the sLeX-negative subpopulation displayed an increased ratio of phosphocholine to glycerophosphocholine when compared to the parental line and sLeX-positive subpopulation. Serum obtained from mice inoculated with either sLeX-negative or sLeX-positive tumor cells contained broader methylene resonances (P = 0.0002; P = 0.0003) and narrower methyl resonances (P = 0.0013; P < 0.0001) when compared to serum of naive mice. However, line widths of methylene and methyl resonances were not useful for distinguishing between the two tumor phenotypes. Results of this study further support the notion that metabolic indicators of malignancy can correlate with in vivo metastatic behavior.