C-13 spectroscopy combined with the injection of C-13-labeled substrates is a powerful method for the study of brain metabolism in vivo. Since highly localized measurements are required in a heterogeneous organ such as the brain, it is of interest to augment the sensitivity of C-13 spectroscopy by proton acquisition. Furthermore, as focal cerebral lesions are often encountered in animal models of disorders in which the two brain hemispheres are compared, we wished to develop a bi-voxel localized sequence for the simultaneous bilateral investigation of rat brain metabolism, with no need for external additional references. Two sequences were developed at 9.4 T: a bi-voxel H-1-(C-13) STEAM-POCE (Proton Observed Carbon Edited) sequence and a bi-voxel H-1-(C-13) PRESS-POCE adiabatically decoupled sequence with Hadamard encoding. Hadamard encoding allows both voxels to be recorded simultaneously, with the same acquisition time as that required for a single voxel. The method was validated in a biological investigation into the neuronal damage and the effect on the Tri Carboxylic Acid cycle in localized excitotoxic lesions. Following an excitotoxic quinolinate-induced localized lesion in the rat cortex and the infusion of U-C-13 glucose, two H-1-(C-13) spectra of distinct (4 x 4 x 4 mm(3)) voxels, one centred on the injured hemisphere and the other on the contralateral hemisphere, were recorded simultaneously. Two H-1 bi-voxel spectra were also recorded and showed a significant decrease in N-acetyl aspartate, and an accumulation of lactate in the ipsilateral hemisphere. The H-1-(C-13) spectra could be recorded dynamically as a function of time, and showed a fall in the glutamatelglutamine ratio and the presence of a stable glutamine pool, with a permanent increase of lactate in the ipsilateral hemisphere. This bi-voxel H-1-(C-13) method can be used to investigate simultaneously both brain hemispheres, and to perform dynamic studies. We report here the neuronal damage and the effect on the Tri Carboxylic Acid cycle in localized excitotoxic lesions.