1. Membrane currents induced by glycine (Iglys) were investigated in cholinergic neurons in primary culture, isolated from the septal region of fetal rat brains, by use of the whole cell voltage-clamp recording configuration. A glycine response was observed in 131 of 150 neurons examined. 2. Igly was accompanied by an increase in conductance and reversed the direction at the Cl- equilibrium potential. The Igly-voltage relation was a little outwardly rectifying in a range of -40 to +30 mV in a nearly symmetrical Cl- solution system. 3. Glycine applied by local perfusion produced an Igly, which increased in amplitude sigmoidally with increasing concentrations (EC50 = 110 microM; nH = 1.75) and desensitized at higher concentrations. There was no nondesensitizing component in Igly (100 microM). Strychnine inhibited Igly in a dose-dependent and competitive manner; it took at most 1 min for this action to be attained to a steady level. Schild analysis yielded the dissociation constant for strychnine to be 0.062 microM. Igly was depressed noncompetitively by a gamma-aminobutyric acid-A (GABAA) receptor-channel blocker, picrotoxin, in a dose-dependent manner (IC50 = 45 microM for peak Igly at 100 microM). On the contrary, another GABAA inhibitor, bicuculline (10 microM), did not affect Igly. 4. Igly was unaffected by spermine (1 mM), ethanol (1-100 mM), and diazepam (1 microM) and was depressed by 14% by pentobarbital (100 microM). On the other hand, Zn2+ potentiated Igly in a dose-dependent (EC50 = 9.1 microM; nH = 1.3; 38% increase for Igly at 50 microM by 100 microM Zn2+) and reversible manner. The facilitatory action was due to an increase in the apparent affinity for glycine (by 139% by 100 microM Zn2+), and did not depend on holding potentials. Pb2+ (100 microM) and La3+ (100 microM) also enhanced Igly (50 microM) by 23 and 14%, respectively; these actions were abolished in the presence of Zn2+ (100 microM). Igly was not affected by Ba2+, Sr2+, Co2+, Mn2+, Cd2+, and Al3+ (100 microM each). 5. Rat septal cholinergic neurons in culture were endowed with a Cl(-)-selective glycine receptor channel whose activation was sensitive to strychnine and picrotoxin. Zn2+ induced a positive modulation of the glycine response, possibly through an allosteric interaction with the glycine-binding site; this action was shared by Pb2+ and La3+ with a potency sequence of Zn2+ > Pb2+ > La3+. This metal-ion binding site could serve to enhance a glycine action.