In order to establish the relative distribution of a GFAP-negative population of astrocytes, and its change in gliotic tissue, sections of the stratum radiatum of the CA1 hippocampal layer of male, adult, Wistar rats were analyzed by immunocytochemical methods. Ten micrometer-thick sections were triple-stained to detect nuclei, glial fibrillary acidic protein (GFAP) and glutamine synthetase (GS). In another set of experiments, the rats received a one-time intraperitoneal injection of kainic acid that caused epileptic seizures. With the use of a behavioral protocol, animals with substantial neuronal loss in the pyramidal layer were selected. Five days after the injection these rats were analyzed similarly to control rats. We find that GFAP-positive cells are a subpopulation of GS-positive cells and that the GFAP-negative subpopulation is quite large (40%). After gliosis the density of GFAP-negative, GS-positive cells stays stable, whereas the GFAP-positive population triples. These experiments confirm electrophysiological experiments showing a distinct, GFAP-negative subset of astrocytes that remains consistent even after injury-induced gliosis and accompanying up-regulation of GFAP.