The (1)H spectrum of certain tumor cells, in vivo tumors, and their biopsies in vitro shows a narrow and intense resonance at 1.26 ppm, which has been assigned to the fatty acyl chain of triglycerides [nuclear magnetic resonance (NMR) visible mobile lipids, MLs]. We have used diffusion-weighted NMR spectroscopy to directly address the subcellular origin of MLs in the case of C6 cells in which lactate accumulation had been inhibited by prior iodoacetamide incubation. Borage oil and artificial lipid droplets were used as model systems of free and restricted diffusion, respectively. The characteristic diameter for the ML resonance compartment measured by NMR for the C6 cells was not significantly different from the one obtained with phase contrast microscopy (1.88 +/- 0.04 micro m from NMR versus 1.37 +/- 0.33 micro m from microscopy). We herewith provide direct and noninvasive evidence that the lipid signal at 1.26 ppm in C6 cells, which remains visible in long echo time (T(E) = 136 ms) experiments, mostly originates from subcellular structures with diameters of 1-2 micro m, which correspond to the cytosolic lipid droplets that can be detected in optical microscopy preparations of the same cells.