Previously we reported that three imaging methods, dynamic contrast enhanced magnetic resonance imaging (DCE-MRI), T1(ρ)-MRI, and ultralow temperature NADH/flavoprotein fluorescence imaging (redox scanning), could differentiate the less metastatic human melanoma cell line A375P from a more metastatic line C8161 growing as mouse xenografts in nude mice (Li LZ et al. Adv. Exp. Med. Biol., 2007, 599:67-78; PNAS, 2009, 106:6608-6613). The more metastatic C8161 tumor was characterized by less blood perfusion/permeability, a more oxidized mitochondrial redox state in the tumor core, and a smaller T1(ρ) relaxation time constant averaged across the entire tumor section. In the current study, we have further probed the bioenergetic status and tissue microenvironment of these tumors by applying whole tumor phosphorous magnetic resonance spectroscopy ((31)P-MRS) to these two xenografts in a vertical bore 9.4-T Varian magnet. The phosphomonoester (PME)/βNTP ratio and intracellular pH value (pHi) were determined. The phosphomonoester (PME)/βNTP was higher in the more metastatic C8161 tumors (n=4) than in the less metastatic A375P tumors (n=4) (p < 0.1). No significant difference between the pHi of C8161 and A375P was observed.