Although we and others have previously shown that newborn central mammalian neurons are more tolerant to anoxia than their adult counterparts, we do not know whether neonatal nerve cells accumulate free cytosolic calcium (Ca2+i) less than adults in response to O2 deprivation. In order to determine whether anoxia increases Ca2+i in adult and neonatal neurons, we monitored calcium in CA1 hippocampal neurons using the calcium-sensitive probe fluo-3 and confocal microscopy. These neurons were studied in the dissociated state in order to study their inherent response to anoxia without the influence of modulatory factors such as synaptic input and neurotransmitters. Severe anoxia caused a rapid increase in Ca2+i in adult CA1 hippocampal neurons, followed by swelling and bleb formation. In neonatal neurons, the latency of this calcium rise was about five times longer than in the adult. Removal of extracellular calcium and addition of calcium channel blockers (Co2+) greatly attenuated the increase in Ca2+i in response to anoxia but did not prevent cell swelling and injury. The addition of glutamate antagonists MK-801 and 6-cyano-7-nitroquinoxaline- 2,3-dione did not affect the increase in Ca2+i induced by anoxia. Replacing extracellular sodium with impermeant cations (N-methyl-D- glucamine) prevented anoxia-induced nerve injury. In addition, Ca2+i levels dropped, rather than increased, during the anoxic period in the absence of sodium; Ca2+i returned toward baseline levels upon reoxygenation. Glutamate was also studied, in the presence of O2, to compare with the effects of anoxia. Adult neurons responded with similar latencies to both anoxia and glutamate (10-500 microM). Neonatal neurons, however, exhibited a more rapid increase in Ca2+i in response to glutamate than they did when exposed to anoxia. We conclude (1) that neonatal neurons increase Ca2+i more slowly than adults in response to anoxia and that this response is due to an influx of Ca2+o into the cytosol, (2) that an increase in Ca2+i is not necessary to induce nerve injury when nerve cells are deprived of O2, (3) that the removal of Nao+ prevents cell swelling and blebbing and is associated with a decrease in Ca2+i during anoxia, and finally (4) that glutamate exposure may not be a completely valid model for the direct action of anoxia on neurons.