13C and 1H nuclear magnetic resonance spectroscopy (NMR) was used to investigate the metabolism of L-lactate and D-glucose in C6 glioma cells. The 13C enrichment of cell metabolites was examined after a 4-h incubation in media containing 5.5 mM glucose and 11 mM lactate, each metabolite being alternatively labelled with either [1-13C]D-glucose or [3-13C]L-lactate. The results indicated that exogenous lactate was the major substrate for oxidative metabolism. They were consistent with the concept of the existence of 2 pools of both lactate and pyruvate, of which 1 pool was closely connected with exogenous lactate and oxidative metabolism, and the other pool was closely related to glycolysis and disconnected from oxidative metabolism. The molecular basis of this behaviour could be related to different locations for the lactate dehydrogenase isoenzymes, as suggested by their immunohistochemical labelling.