Glycine is an inhibitory neurotransmitter activating a chloride conductance in the mammalian CNS. In vitro studies from brain slices revealed a novel presynaptic site of glycine action in the medial nucleus of the trapezoid body (MNTB) which increases the release of the excitatory transmitter glutamate from the calyx of Held. Here, we investigate the action of glycine on action potential firing of single MNTB neurons from the gerbil under acoustic stimulation in vivo. Iontophoretic application of the glycine receptor antagonist strychnine caused a significant decrease in spontaneous and sound-evoked firing rates throughout the neurons' excitatory response areas, with the largest changes at the respective characteristic frequency (CF). The decreased firing rate was accompanied by longer and more variable onset latencies of sound-evoked responses. Outside the neurons' excitatory response areas, firing rates increased during the application of strychnine due to a reduction of inhibitory sidebands, causing a broadening of frequency tuning. These results indicate that glycine enhances the efficacy for on-CF stimuli, while simultaneously suppressing synaptic transmission for off-CF stimuli. These in vivo results provide evidence of multiple excitatory and inhibitory glycine effects on the same neuronal population in the mature mammalian CNS.