Calcium imaging and patch-clamp recording techniques were used to investigate the relationship between membrane properties and intracellular calcium changes in response to the excitatory amino acid neurotransmitter glutamate. Application of glutamate to cultured neocortical neurons produced concentration-dependent increases in intracellular calcium, membrane depolarization and transmembrane current. At a low concentration (3 μM), glutamate induced only a small depolarization ( < 10 mV), yet produced a substantial increase in intracellular calcium. The calcium increase was observed in the presence of extracellular magnesium, was dependent on extracellular calcium, was blocked by an N-methyl-d-aspartate receptor antagonist, and was not affected by manipulation of intracellular calcium stores. This low concentration of glutamate also induced membrane currents that exhibited an N-methyl-d-aspartate-like unconventional voltage dependence. When glutamate was increased to a concentration known to produce excitotoxicity (500 μM), large depolarizations and membrane currents were induced, which rapidly reversed following prolonged glutamate applications. Changes in intracellular calcium in response to 500 μM glutamate had both voltage-sensitive and -insensitive components, and consistently remained elevated following removal of glutamate. These results indicate that low concentrations of glutamate can preferentially activate N-methyl-d-aspartate receptors, leading to increases in intracellular calcium. Functionally this may be involved in N-methyl-d-aspartate receptor responses to ambient extracellular glutamate. In addition, N-methyl-d-aspartate receptor-mediated calcium influx and subsequent depolarization induced by high glutamate concentrations can produce alterations in intracellular calcium homeostasis, which may play an important role in excitotoxicity. © 1995 IBRO.