Previous studies in our laboratories found that isolectin B(4)(IB(4))-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. Considering that IB(4)-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 IB(4) was injected into the nerves innervating quadriceps muscle (femoral) or hindlimb skin (saphenous) of male C57Bl/6 mice. Similarly, Alexa Fluor-conjugated cholera toxin-beta 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 IB(4) 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 IB(4)). In contrast, NHF staining was more prevalent among femoral afferents, followed by saphenous (WGA) and colon afferents. IB(4) binding was observed in very few colon or saphenous (WGA) afferents, with no femoral afferents binding or transporting IB(4). 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.