1 Departments of Otolaryngology—Head and Neck Surgery, Neurobiology and Anatomy and the Sensory Neuroscience Research Center, West Virginia University School of Medicine, Morgantown, West Virginia; and 2 Auditory Research Center, Lake Erie College of Osteopathic Medicine, Erie, Pennsylvania Submitted 13 June 2006; accepted in final form 15 November 2006 The superior paraolivary nucleus (SPON) is a prominent periolivary cell group of the superior olivary complex. SPON neurons use -aminobutyric acid (GABA) as their neurotransmitter and are contacted by large numbers of glycinergic and GABAergic punctate profiles, representing a dense inhibitory innervation from the medial nucleus of the trapezoid body (MNTB) and from collaterals of SPON axons, respectively. SPON neurons have low rates of spontaneous activity, respond preferentially to the offset of pure tones, and phase-lock to amplitude-modulated tones. To determine the roles of glycine and GABA in shaping SPON responses, we recorded from single units in the SPON of anesthetized rats before, during, and after application of the glycine receptor antagonist strychnine, the GABA A receptor antagonist bicuculline, or both drugs applied simultaneously. Strychnine caused a major increase in spike counts during the stimulus presentation, followed by the disappearance of offset spikes. In half of the recorded units, bicuculline caused moderately increased firing during the stimulus. However, in 86% of units bicuculline also caused a large increase in the magnitude of the offset response. Application of the drug cocktail caused increased spontaneous activity, dramatically increased spike counts during the stimulus presentation, and eliminated the offset response in most units. We conclude that glycinergic inhibition from the MNTB suppresses SPON spiking during sound stimulation and is essential in generating offset responses. GABAergic inhibition, presumably from intrinsic SPON collaterals, plays a subtler role, contributing in some cells to suppression of firing during the stimulus and in most cells to restrict firing after stimulus offset. Address for reprint requests and other correspondence: A. Berrebi, Sensory Neuroscience Research Center, PO Box 9303, Health Sciences Center, West Virginia University School of Medicine, Morgantown, WV 26506-9303 (E-mail: aberrebi{at}hsc.wvu.edu )