The purpose of the present study was to examine the effect of blockade of N-methyl-D-aspartate (NMDA) receptors on the depolarization associated with severe hypoglycemia, which is commonly preceded by one or a few transient depolarizations reminiscent of cortical spreading depression (CSD). In the cerebral cortices of rats [K⁺](e) and [Ca²⁺](e) were measured with ion-selective microelectrodes. NMDA blockade was achieved by injection of MK801 in doses that block CSD. In control rats, the latency from the time point when blood glucose reached minimal levels to onset of ionic shifts was 33.2 ± 3.5 min, and [K⁺](e) rose from 3.2 ± 0.2 to 55 ± 5 mM. All variables remained unchanged in rats treated with MK801. In another four rats treated with MK801, [Ca²⁺](e) declined from 1.06 ± 0.22 to 0.12 ± 0.02 mM. Plasma glucose measurements indicated that the cortex depolarized at a plasma glucose concentration between 0.7 and 0.8 mM, i.e., within a narrow range, suggesting a threshold phenomenon. In conclusion, activation of NMDA receptors seems of minor importance for hypoglycemic depolarization. The ionic transients that precede the persistent hypoglycemic depolarization are probably mediated by mechanisms distinct from those of electrically induced CSD.