A predictable pattern of selective neuronal cell death occurs in organotypic slice cultures of neonatal rat hippocampus during the second and third weeks in vitro. We serially examined organotypic cultures at three, four, seven, 14, 21 and 28 days in vitro, using uptake of the fluorescent dye propidium iodide to identify degenerating cells. After seven days in vitro, the cell degeneration that accompanies the slicing procedure appears to have ended. However, at 14 days in vitro, degenerating neurons could be identified in area CA3. When many degenerating cells were present in a slice, they were distributed in the dentate hilus (CA4) and proximal portions of CA1 as well. Neuronal degeneration involving mainly CA1 pyramidal cells was still apparent at 21 days in vitro, but was much less marked than at 14 days. Study of fixed cultures with light and electron microscopy methods confirmed the presence of degenerating neurons with a pyknotic or vacuolated appearance. Spontaneous neuronal degeneration at 14 and at 21 days in vitro was almost entirely prevented by the addition of 10.5 mM Mg2+ or 3 mM kynurenic acid (a glutamate receptor antagonist), beginning at seven days in vitro. Cell death was markedly decreased by treatment with 100μM dl-2-amino-5-phosphonovaleric acid (a selective antagonist of N-methyl-d-aspartate glutamate receptors). Removal of the blocking agents by returning cultures to control media at 28 days in vitro induced widespread neuronal degeneration, involving all the regions of the hippocampal slice cultures. The inhibition of spontaneous neuronal cell death by glutamate receptor antagonists and by blockade of glutamate release at synapses suggests that the mechanism of cell death involves glutamate receptors. The time course of degeneration suggests that the vulnerability to glutamate excitotoxicity is an aspect of developmentally regulated components of glutamatergic synapses acquired in the hippocampal organotypic cultures after the first week in vitro. © 1994.