The aim of these investigations was to examine directly with fura-2 microspectrofluorimetry, the effects of general anesthetics on the resting level and glutamate-stimulated increase of intraneuronal free calcium ([Ca++]i) in cultured hippocampal neurons. Media were chosen for the preferential activation by glutamate of either the quisqualate (QUIS media) or N-methyl-D-aspartate (NMDA media) receptor subtypes. Continuous perfusion (20 min) of either media that had been saturated with isoflurane (0.5-4%) or, in some cases halothane (3-4%), produced only small and inconsistent changes in resting [Ca++]i. The rise in [Ca++]i induced by glutamate (or in some cases, NMDA) that was applied in the mainstream of QUIS or NMDA media was attenuated greatly during such applications of isoflurane or halothane for 6 to 20 min. Analysis of concentration-response relationships revealed that the EC50 values for the isoflurane-depressions were 1.7% for the Ca response to glutamate in QUIS media and 1.2% in NMDA media. Application of isoflurane blunted the peak increases in [Ca++]i produced by brief (1 min) applications of 50 mM K. Verapamil (25 microM) did not reduce the resting [Ca++]i and had long-lasting depressant effects on glutamate-stimulated increases in [Ca++]i in NMDA media. The effects of 2% isoflurane and verapamil were approximately additive. These investigations provided evidence that isoflurane reduced the increase in [Ca++]i which resulted from Ca influx linked directly to receptors for glutamate in addition to Ca entry due to activation of voltage-gated Ca channels.