Previous studies in our laboratories found that isolectin B₄(IB₄)-positive polymodal nociceptors in the mouse do not express transient receptor potential vanilloid 1 (TRPV1), nor does deletion of TRPV1 compromise the ability of these afferents to detect thermal stimuli [Zwick M, Davis BM, Woodbury CJ, Burkett JN, Koerber HR, Simpson JF, Albers KM (2002) Glial cell line-derived neurotrophic factor is a survival factor for isolectin B4-positive, but not vanilloid receptor 1-positive, neurons in the mouse. J Neurosci 22:4057–4065; Woodbury CJ, Zwick M, Wang S, Lawson JJ, Caterina MJ, Koltzenburg M, Albers KM, Koerber HR, Davis BM (2004) Nociceptors lacking TRPV1 and TRPV2 have normal heat responses. J Neurosci 24:6410–6415]. Considering that IB₄-positive afferents account for over 70% of cutaneous nociceptors and that 30–50% of all mouse primary afferents express TRPV1, it is highly likely that many TRPV1-positive fibers project to non-cutaneous structures. To investigate this issue, Alexa Fluor-conjugated wheat germ agglutinin (WGA) or IB₄ was injected into the nerves innervating quadriceps muscle (femoral) or hindlimb skin (saphenous) of male C57Bl/6 mice. Similarly, Alexa Fluor-conjugated cholera toxin-β was injected subserosally into the distal colon. Spinal ganglia at the appropriate level (L2–3 for saphenous and femoral nerves; L6 for colon) were processed for TRPV1, calcitonin gene-related peptide (CGRP), neurofilament heavy chain (NHF) and IB₄ visualization and examined on a confocal microscope. Colon afferents contained the highest percentage of both TRPV1- and CGRP-positive neurons, followed by femoral (WGA) and saphenous afferents (WGA and IB₄). In contrast, NHF staining was more prevalent among femoral afferents, followed by saphenous (WGA) and colon afferents. IB₄ binding was observed in very few colon or saphenous (WGA) afferents, with no femoral afferents binding or transporting IB₄. Considering that the largest percentages of TRPV1-positive neurons observed in this study were within visceral and muscle afferent populations (neurons that typically are not subject to noxious temperatures), these results suggest that TRPV1 may not function primarily as a temperature sensor but rather as a detector of protons, vanilloid compounds or through interactions with other membrane proteins.