Three vesicular glutamate transporters (VGLUTs) have been recently identified and their distribution has been mapped in various brain areas. In the present study, we used morphological approaches to investigate their expression in the rat lumbar spinal cord and dorsal root ganglia. Our results show a complementary distribution of VGLUT-expressing fibers in the spinal cord, with no overlapping in nerve endings. In the dorsal horn, VGLUT1 is most abundant in mechanosensory/proprioceptive deep afferent fibers. VGLUT2 and VGLUT3 are expressed only at moderate levels in primary sensory afferent fibers and are not used by central projections of nociceptive neurons. VGLUT1 and VGLUT2 mRNAs are mainly segregated in superficial laminae but colocalized in deeper laminae. Weak expression of VGLUT3 mRNA is only detected in deep laminae. The colocalization of VGLUT1 and VGLUT2 transcripts in most sensory neurons of the dorsal root ganglia is not in agreement with the clear segregation between the proteins in their spinal projections. Such a discrepancy suggests targeting mechanisms specific for each transporter and/or a distinct regulation of their translation. In the ventral horn, the expression of VGLUT1 and VGLUT2 mRNAs in motoneuron perikarya suggests the possible unexpected role of glutamate in the vertebrate neuromuscular junction. These results demonstrate the existence of different subpopulations of glutamate nerve terminals in the rat lumbar spinal cord and suggest that functionally distinct subsets of excitatory glutamatergic neuronal networks are involved in sensory processing and motor control. J. Comp. Neurol. 468:380–394, 2004. © 2003 Wiley-Liss, Inc.