The time course of currents mediated by native and recombinant glycine receptors was examined with a combination of rapid agonist applications to outside-out patches and single-channel recording. The deactivation time constant of currents evoked by brief, saturating pulses of glycine is profoundly affected by the chloride concentration on the intracellular side of the cell membrane. Deactivation was threefold slower when intracellular chloride was increased from a low level (10 mm), similar to that observed in living mature neurons, to 131 mm ("symmetrical" chloride, often used in pipette internal solutions). Single-channel analysis revealed that high chloride has its greatest effect on the channel closing rate, slowing it by a factor of 2 compared with the value we estimated in the cell-attached mode (in which the channels are at physiological intracellular chloride concentrations). The same effect of chloride was observed when glycinergic evoked synaptic currents were recorded from juvenile rat spinal motoneurons in vitro, because the decay time constant was reduced from approximately 7 ms to approximately 3 ms when cells were dialyzed with 10 mm chloride intracellular recording solution. Our results indicate that the time course of glycinergic synaptic inhibition in intact neurons is much faster than is estimated by measurements in symmetrical chloride and can be modulated by changes in intracellular chloride concentration in the range that can occur in physiological or pathological conditions.