Recent studies have demonstrated that several specific types of acoustically-evoked GABA-mediated inhibition occur in neurons of the central nucleus of inferior colliculus (ICc). The dorsal nucleus of the lateral lemniscus (DNLL) provides a major GABAergic projection to ICc. The present study examined the effects of electrical or chemical stimulation or reversible blockade within the DNLL on the discharge characteristics of ICc neurons in anesthetized rats. Microinjection of a local anesthetic (lidocaine) or a GABA-A agonist (THIP) via a cannula placed into DNLL reversibly blocked acoustically-evoked binaural inhibition and increased spontaneous firing in most contralateral ICc neurons. Trains of electrical pulses or microinjection of the excitant amino acid, kainate, into DNLL resulted in reduced acoustically-evoked firing, which was similar to binaural inhibition, in most contralateral ICc neurons examined. The effects of DNLL electrical stimulation were reversibly blocked by microinjection of THIP into the stimulation site, suggesting that the effect of the electrical stimulation is mediated by direct effects on cell bodies of DNLL neurons. These data support the idea that contralateral GABAergic input from the DNLL is inhibitory to ICc neurons. Thus, binaural inhibition and tonic inhibition in ICc neurons may be mediated, in part, by the GABAergic projection from the contralateral DNLL.