Projections of glutamatergic somatosensory and auditory fibers to the cochlear nucleus (CN) are mostly nonoverlapping: projections from the spinal trigeminal nucleus (Sp5) terminate primarily in the granule cell domains (GCD) of CN, whereas type I auditory nerve fibers (ANFs) project to the magnocellular areas of the VCN (VCNm) and deep layers of Dorsal CN (DCN). Vesicular glutamate transporters (VGLUTs), which selectively package glutamate into synaptic vesicles, have different isoforms associated with distinct subtypes of excitatory glutamatergic neurons. Here we examined the distributions of VGLUT1 and VGLU2 expression in the CN and their colocalization with Sp5 and ANF terminals following injections of anterograde tracers into Sp5 and the cochlea in the guinea pig. The CN regions that showed the most intense expression of VGLUT1 and VGLUT2 were largely nonoverlapping and were consistent with ANF and Sp5 projections, respectively: VGLUT1 was highly expressed in VCNm and the molecular layer of the DCN, whereas VGLUT2 was expressed predominantly in the GCD. Half (47% ± 3%) of the Sp5 mossy fiber endings colabeled with VGLUT2, but few (2.5% ± 1%) colabeled with VGLUT1. In contrast, ANFs colabeled predominantly with VGLUT1. The pathway-specific expression of VGLUT isoforms in the CN may be associated with the intrinsic synaptic properties that are unique to each sensory pathway. J. Comp. Neurol. 500:777–787, 2007. © 2006 Wiley-Liss, Inc.