Antisera were raised in rabbits against aspartate or glutamate conjugated to bovine serum albumin by glutaraldehyde. After immunosorbent purification the antisera reacted selectively with brain protein-glutaraldehyde conjugates of the respective amino acids. These results from model systems encouraged us to employ the antisera to study the distribution of free aspartate and glutamate in brain tissue. The aspartate antiserum produced intense staining of interneurons and deep hilar neurons and modest labelling of pyramidal and granular cells in the hippocampal formation of rats, guinea-pigs and baboons perfusion-fixed with glutaraldehyde. In contrast, glutamate-like immunoreactivity was generally high in pyramidal and granular cells and low in interneurons. In hippocampal slices immersion fixed in glutaraldehyde after being soaked in Krebs' solution aspartate-like and glutamate-like immunoreactivities were lost from perikarya and dendrites. The staining that remained occurred in nerve terminal-like dots and matched the distribution of the major excitatory fiber systems, except that only glutamate-like immunoreactivity, and not aspartate-like immunoreactivity, was concentrated at the site of the mossy fiber terminals, and that aspartate-like but not glutamate-like immunoreactivity occurred between the granular and pyramidal cell bodies. The present technique specifically demonstrates aspartate and glutamate in glutaraldehyde-fixed tissue. We suggest that in perfusion-fixed material the staining intensities reflect the total concentrations of the amino acids (i.e. the "metabolic pool" plus the "transmitter pool"). In immersion-fixed hippocampal slices the "transmitter pool" may be preferentially visualized.