Activation of unmyelinated (C-fiber) nociceptors by noxious chemicals plays a critical role in the initiation and maintenance of injury-induced pain. C-fiber nociceptors can be divided into two groups in which one class depends on nerve growth factor during postnatal development and contains neuropeptides, and the second class depends on glial cell line-derived neurotrophic factor during postnatal development and contains few neuropeptides but binds isolectin B4 (IB4). We determined the sensitivity of these two populations to protons and capsaicin using whole cell recordings of dorsal root ganglion neurons from adult mouse. IB4-negative unmyelinated neurons were significantly more responsive to protons than IB4-positive neurons in a concentration-dependent manner. Approximately 86% of IB4-negative neurons responded to pH 5.0 with an inward current compared with only 33% of IB4-positive neurons. The subtypes of proton-evoked currents in IB4-negative unmyelinated neurons were also more diverse. Many IB4-negative neurons exhibited transient, rapidly inactivating proton currents as well as sustained proton currents. In contrast, IB4-positive neurons never displayed transient proton currents and responded to protons only with sustained, slowly inactivating inward currents. The two classes of neurons also responded differently to capsaicin. Twice as many naı̈ve IB4-negative unmyelinated neurons responded to 1 μM capsaicin as IB4-positive neurons, and the capsaicin-evoked currents in IB4-negative neurons were approximately fourfold larger than those in IB4-positive neurons. Interestingly, proton exposure altered the capsaicin responsiveness of the two classes of neurons in opposite ways. Brief preexposure to protons increased the number of capsaicin-responsive IB4-positive neurons by twofold and increased the capsaicin-evoked currents by threefold. Conversely, proton exposure decreased the number of capsaicin-responsive IB4-negative neurons by 50%. These data suggest that IB4-negative unmyelinated nociceptors are initially the primary responders to both protons and capsaicin, but IB4-positive nociceptors have a unique capacity to be sensitized by protons to capsaicin-receptor agonists.