The inferior colliculus is a major relay nucleus in the ascending auditory pathways that receives multiple glutamatergic inputs. Vesicular glutamate transporters 1 and 2 (VGLUT1, VGLUT2) most often have complementary non-overlapping distributions and can be used to differentiate glutamatergic inputs. The present study therefore examined co-immunolabeling of VGLUT1 and VGLUT2 in three divisions of the inferior colliculus. Additional co-immunolabeling of microtubule associated protein 2 and neuronal class III β-tubulin provided visualization of neuronal soma and processes and allowed identification of axo-somatic versus axo-dendritic contacts. Results showed numerous VGLUT1 and 2 immunolabeled terminals in the central nucleus, lateral cortex and dorsal cortex. In all three divisions there was little to no co-containment of the two vesicular glutamate transporters indicating a complementary distribution. VGLUT1 made predominantly axo-dendritic connections in the neuropil, while VGLUT2 had many axo-somatic contacts in addition to axodendritic contacts. VGLUT2 immunolabeled terminals were numerous on the soma and proximal dendrites of many medium-to-large and large neurons in the central nucleus and medium to large neurons in the dorsal cortex. There were more VGLUT2 terminals than VGLUT 1 in all divisions and more VGLUT2 terminals in dorsal and lateral cortices than in the central nucleus. This study shows that VGLUT1 and VGLUT2 differentiate complementary patterns of glutamatergic inputs into the central nucleus, lateral and dorsal cortex of the inferior colliculus with VGLUT1 endings VGLUT1 endings predominantly on the dendrites and VGLUT2 on both dendrites and somas. Keywords auditory; auditory brain stem; co-localization Glutamate is the major excitatory transmitter in the ascending auditory pathway, used by multiple neurons and multiple projections at all the different levels. The presence of vesicular glutamate transporters (VGLUTs) which mediate the packaging of the excitatory neurotransmitter glutamate into synaptic vesicles has proven to be a useful immunocytochemical marker for glutamatergic terminals. There are at least three subtypes of VGLUTs: VGLUT1, VGLUT2 and VGLUT3. VGLUT1 and VGLUT2 are both associated with glutamatergic terminals and their localizations are generally complementary without overlap (e.g. Hioki et al, 2003; The inferior colliculus (IC) receives multiple inputs from ascending and descending auditory pathways as well as from non-auditory inputs (Adams 1979, 1980, 1983, Aitkin et al, 1981Bajo and Moore, 2005;Cant & Benson, 2006;Jain & Shore, 2006;Loftus et al, 2004;Malmierca et al, 2005;Marsh et al, 2002;Merchan et al, 1994;Moore et al, 1998; Okayama et al, 2006;Oliver, 2000;Oliver et al, 1997; Osen, 1972;Saldana et al, 1996; Shofield, 2002; Shofield and Cant, 1996; Zhou, 2006, Tokunaga et al, 1984;Zhou and Shore, 2006) many of which are glutamatergic (Malmierca, 2003 for review;Alibardi, 1998;Feliciano and Potashner, 1995;Ross et al, 1995;Saint Marie, 1996, Suneja et al, 1995....