The light microscopic localization of aspartate-like immunoreactivity (Asp-LI) was compared to that of glutamate-like immunoreactivity (Glu-LI) in hippocampal slices by means of specific polyclonal antibodies recognizing the amino acids fixed by glutaraldehyde. After incubation in Krebs' solution with normal (5 mM) or depolarizing concentrations of K+, and various additives, the slices were fixed with glutaraldehyde, resectioned and processed according to the peroxidase - antiperoxidase procedure. At 5 mM K+, Glu-LI was localized in nerve-terminal like dots with a conspicuous laminar distribution, the highest Glu-LI concentrations coinciding with the terminal fields of major excitatory pathways thought to use glutamate or aspartate as transmitters. The localization of Asp-LI showed some similarity to that of Glu-LI, but the laminar distribution was less differentiated and the immunoreactivity was much weaker. At 40 and 55 mM K+ the nerve terminal localizations of Glu-LI and Asp-LI were strongly reduced. Concomitantly, both immunoreactivities appeared in astroglial cells. These changes were Ca2+-dependent. The nerve ending staining patterns of Asp-LI and Glu-LI could be sustained during depolarization if the medium was supplemented with glutamine (0.5 mM). Under these conditions Asp-LI became more intense and its distribution approached that of Glu-LI. This suggests that, when stimulated, some nerve endings can increase their reservoir of releasable aspartate. The presence of glutamine during depolarization strongly reduced glial Asp-LI and Glu-LI, possibly due to its providing nitrogen for conversion of glutamate to glutamine. alpha-Ketoglutarate, another glia-derived precursor of neuronal glutamate, was virtually ineffective in supporting Glu-LI and Asp-LI in nerve endings, and did not suppress Glu-LI or Asp-LI in glia. Our findings provide morphological support for the view that excitatory nerve endings under certain conditions can contain high levels of both aspartate and glutamate (possibly in the same terminals), and that aspartate as well as glutamate can be released synaptically. Further, they underline the importance of the glial supply of the nerve endings with precursor glutamine, which allows them to build up and sustain high concentrations of transmitter amino acids during release.