In vertebrates, most glycinergic inhibitory neurons discharge phasically at a relatively low frequency. Such a pattern of glycine liberation from presynaptic terminals may affect the kinetics of post-synaptic glycine receptors. To examine this influence, we have analyzed the behavior of glycine receptors in response to repetitive stimulation at frequencies at which consecutive outside-out currents did not superimpose (0.5-4 Hz). Neurotransmitter release was mimicked on outside-out patches from zebrafish hindbrain Mauthner cells using fast flow application techniques. The amplitude of outside-out currents evoked by short (1 ms) repetitive applications of a saturating concentration (3 mM) of glycine remained unchanged for application frequencies<or=1 Hz. When the application frequency was increased from 1 to 4 Hz, the amplitude of the outside-out currents decreased with time to reach a steady state level. This decrease in current amplitude was larger and occurred faster with increasing application frequencies. Recovery occurred when the stimulation frequency was decreased back to 1 Hz. The recovery time constant was independent on the application frequency. This frequency-dependent inhibition was also observed for non-saturating glycine concentrations. Our results indicate that glycine receptor activity is down-regulated when the stimulation frequency increases to values>1 Hz. Glycine-evoked current simulations using a simple Markov model describing zebrafish glycine receptor kinetic behavior, indicates that this down-regulation of glycine receptor efficacy is due to a progressive accumulation of the receptors in a long lasting desensitization state. Our simulations suggest that this down-regulation can occur even when spontaneous inhibitory currents were generated randomly at a frequency>1 Hz.