Although glia are known to be sensitive to exogenously applied neurotransmitter substances, little is known about how glia respond to neuronal activity on the millisecond time scale of individual synaptic events. We have explored the electrophysiological effects of excitatory neuronal signaling on glial cells in rat hippocampal microcultures. Exogenous applications of glutamate analogs to islands of glia revealed a large ionotropic AMPA receptor-mediated current and a smaller current mediated by electrogenic glutamate uptake. Glia demonstrated no evidence of NMDA or metabotropic receptor-mediated currents or membrane conductance changes. Despite the dominant contribution of AMPA receptors to exogenous glutamate applications in glia, AMPA receptor currents contributed only approximately 20% to the response of glia to endogenous glutamate release from solitary excitatory neurons. Electrogenic glutamate uptake contributed strongly to glial responses to neuronal stimulation. In addition, some glia exhibited a residual current is response to neuronal stimulation that was not attributable to calcium-dependent transmitter release or to neuronal potassium efflux. These results help provide a context for understanding the role of glial transporters and receptors in nervous system signaling.