Although it is well documented that glutamate receptor subtypes are differentially expressed during central nervous system development postnatally, how glutamate affects neurons during postnatal development is unclear. We therefore examined the development of the intrinsic neuronal response to glutamate receptor activation by studying single, hippocampal CA1 neurons that had been acutely dissociated from newborn (P1-3), 1 week old (P6-8), and 3 week old (P21-25) rats. Using laser scanning confocal microscopy and the calcium dye Fluo-3, we made time-lapse studies of the effects of glutamate stimulation on free intracellular calcium ([Ca2+]i) and simultaneous changes in neuronal morphology. In P21-25 neurons, glutamate increased [Ca2+]i fluorescence, and caused marked somal swelling, blebbing, and retraction of dendrites into the soma. These major morphological changes were followed by sudden loss of intracellular fluorescence, indicative of a loss of membrane integrity and cell death. In P6-8 neurons, glutamate increased [Ca2+]i to the same extent, but this increase was not followed by either major morphological changes or loss of membrane integrity. In P1-3 neurons, glutamate increased [Ca2+]i minimally, and no morphologic changes were observed. P1-3 neurons dissociated without enzymatic digestion demonstrated glutamate responses identical to responses seen in neurons dissociated with enzymatic digestion. In the presence of MK-801 (15 microM), glutamate still increased [Ca2+]i and caused cell death in P21-25 neurons, but the latency to these effects more than tripled. This late, MK-801-resistant [Ca2+]i increase was not eliminated by DNQX or Ni2+/Cd2+, suggesting that this increase is mediated by metabotropic receptors. These findings demonstrate that (1) hippocampal neurons from newborns are intrinsically less vulnerable to glutamate toxicity than neurons from 3 weeks old animals, and (2) multiple glutamate receptor subtypes affect the magnitude of the [Ca2+]i increase in response to glutamate in the neuronal microenvironment.