The vanilloid receptor-like 1 protein (VRL-1, also called TRPV2) is a member of the TRPV family of proteins and is a homolog of the capsaicin/vanilloid receptor (VR1, or TRPV1). Although VRL-1 does not bind capsaicin, like VR1 it is activated by noxious heat (>52°C). Unlike VR1, however, VRL-1 is primarily expressed by medium- and large-diameter primary afferents, which suggests that nociceptive processing is but one of the functions to which VRL-1 contributes. To provide information on the diverse spinal circuits that are engaged by these VRL-1-expressing primary afferents, we completed a detailed immunocytochemical map of VRL-1 in rat spinal cord, including light and electron microscopic analysis, and generated a more comprehensive neurochemical characterization of VRL-1-expressing primary afferents. Consistent with previous reports, we found that VRL-1 and VR1 are expressed in different dorsal root ganglion (DRG) cell bodies. Almost all VRL-1-expressing cells labeled for N52 (a marker of myelinated afferents), consistent with VRL-1 expression in Aδ and Aβ fibers. EM analysis of the DRG and dorsal roots confirmed this and revealed two categories of neurons based on the intensity of immunolabeling. The densest VRL-1 immunoreactivity in the spinal cord was found in lamina I, inner lamina II, and laminae III/IV. This is consistent with the expression of VRL-1 by myelinated nociceptors that target laminae I and IIi and in nonnociceptive Aβ fibers that target laminae III/IV. Dorsal rhizotomy reduced, but did not eliminate, the immunostaining in all dorsal horn laminae, which indicates that VRL-1 expression derives from both DRG cells and from neurons intrinsic to the brain or spinal cord. Spinal hemisection reduced immunostaining of the ipsilateral dorsal columns in segments rostral to the lesion and in the dorsal column nuclei, presumably from the loss of ascending Aβ afferents, but there was no change caudal to the lesion. Thus, supraspinal sources of dorsal horn VRL-1 immunoreactivity are likely not significant. Although we never observed VRL-1 immunostaining in cell bodies in the superficial dorsal horn, there was extensive labeling of motoneurons and ventral root efferents—in particular, in an extremely densely labeled population at the lumbosacral junction. Finally, many ependymal cells surrounding the central canal were intensely labeled. These results emphasize that VRL-1, in contrast to VR1, is present in a diverse population of neurons and undoubtedly contributes to numerous functions in addition to nociceptive processing. J. Comp. Neurol. 470:400–408, 2004. © 2004 Wiley-Liss, Inc.