There is evidence from immunohistochemistry, quantitative microchemistry, and pharmacology for several amino acids as neurotransmitters in the vestibular nuclear complex (VNC), including glutamate, γ-aminobutyrate (GABA), and glycine. However, evidence from measurements of release has been limited. The purpose of this study was to measure depolarization-stimulated calcium-dependent release of amino acids from the VNC in brain slices. Coronal slices containing predominantly the VNC were prepared from rats and perfused with artificial cerebrospinal fluid (ACSF) in an interface chamber. Fluid was collected from the chamber just downstream from the VNC using a microsiphon. Depolarization was induced by 50 mM potassium in either control calcium and magnesium concentrations or reduced calcium and elevated magnesium. Amino acid concentrations in effluent fluid were measured by high performance liquid chromatography. Glutamate release increased fivefold during depolarization in control calcium concentration and twofold in low calcium/high magnesium. These same ratios were 6 and 1.5 for GABA, 2 and 1.3 for glycine, and 2 and 1.5 for aspartate. Differences between release in control and low calcium/high magnesium ACSF were statistically significant for glutamate, GABA, and glycine. Glutamine release decreased during and after depolarization, and taurine release slowly increased. No evidence for calcium-dependent release was found for serine, glutamine, alanine, threonine, arginine, taurine, or tyrosine. Our results support glutamate and GABA as major neurotransmitters in the VNC. They also support glycine as a neurotransmitter and some function for taurine.