Hyperpolarizing responses in neuropil glial cells evoked by nerve root stimulation were studied in the central nervous system of the leech Hirudo medicinalis using intracellular recording and extracellular stimulation techniques. From a mean resting potential of -60.5 +/- 1.0, the glial membrane was hyperpolarized by -8.6 +/- 0.8 mV, via stimulation of the dorsal posterior nerve root in an isolated ganglion. Nerve root stimulation evoked biphasic or depolarizing responses in glial cells with resting potentials around -70 mV (Rose CR, Deitmer JW. J. Neurophysiol. 73:125-131, 1995). The hyperpolarizing response was reduced by the ionotropic glutamate receptor antagonist CNQX (50 microM) to 58% of its initial amplitude. In 15 mM Ca2+/15 mM Mg(2+)-saline the hyperpolarization was reduced by 44%. The hyperpolarization that persisted in high-divalent cation saline was not affected by CNQX. Bath-applied glutamate (500 microM) and kainate (2 microM) elicited glial hyperpolarizations that were sensitive to CNQX and 10 mM Mg2+/1 mM Ca(2+)-saline. The 5-HT-antagonist methysergide did not affect the hyperpolarizations evoked by nerve root stimulation. The results show that in the leech glial membrane responses to neuronal activity include not only depolarizations, as shown previously, but also hyperpolarizations, which are mediated by direct and indirect neuron-glial communication pathways. In the indirect pathway, glutamate is a transmitter between neurons.