Antisera raised against protein-glutaraldehyde-amino acid conjugates were used to study the light and electron microscopic distribution of GABA, glutamate, glutamine and taurine in organotypic slice cultures of rat hippocampi. In the stratum oriens and radiatum, glutamate-like immunoreactivity was particularly concentrated in nerve endings establishing asymmetric junctions with dendritic spines. Mossy fiber terminals in CA3 and the dentate hilus were also strongly labeled. A quantitative immunogold analysis of the glutamate-immunolabelled profiles showed a pattern that was highly reminiscent of that previously observed in perfusion-fixed hippocampi, including a correspondingly sparse labeling of glial processes and of presynaptic elements in symmetric synapses. GABA-like immunoreactivity was localized predominantly in interneurons and in presynaptic terminals contacting dendritic shafts and neuronal cell bodies, while immunoreactivities for glutamine and taurine were found mainly in astroglial cells and pyramidal cells, respectively. Our data indicate that the major intrinsic fiber systems of the cultured hippocampi have retained their normal transmitter phenotypes.