This study characterized the excitatory (nociceptive) and desensitizing (antinociceptive) properties of the natural pungent substances, capsaicin (CAP) and resiniferatoxin (RTX) instilled in the bladder (intravesical, i.ves.) via an indwelling cannula in awake, freely moving rats. The incidence of 9 behaviors was scored for 10 min following i.ves. vehicle or RTX (1.0 nmol). Abdominal licking and head-turning occurred significantly more often in RTX-treated rats compared to vehicle controls, whereas head-grooming, locomotion, rearing and biting did not differ between the two groups. Little or no vocalization, defecation or hindlimb hyperextension was observed in either RTX- or vehicle-treated rats. A second injection of either vehicle or RTX administered to RTX-treated rats 60 min later did not significantly increase abdominal licking or head-turning compared to vehicle controls; this subsequent lack of excitation was taken as a measure of desensitization. In a separate experiment, the first injection of i.ves. RTX (0.1–3.0 nmol) increased licking in a dose-dependent manner; in contrast, the first injection of i.ves. CAP (0.1–3.0 μmol) significantly increased licking only at the intermediate dose tested, 1.0 μmol. With each subsequent injection of the same drug and dose at 30-min intervals, licking increased to a lesser extent, such that it was not significantly different from control after the fourth injection. Rats treated with i.ves. CAP or RTX also did not show increased licking when administered the opposite treatment 30 min later (RTX or CAP, respectively), indicating cross-desensitization; however, i.ves. administration of a third, higher dose of RTX reinstated licking behavior in these ‘desensitized’ rats. Subcutaneous administration of CAP (18–180 mg/kg) or RTX (18–180 μg/kg) dose-dependently attenuated the excitatory response to i.ves. CAP and RTX administered 2 days later. Whereas rats treated systemically with RTX also were desensitized to the excitatory effects of RTX instilled in the eye (evaluated in the eye-wipe assay), rats treated i.ves. with RTX and vehicle-treated rats showed a normal eye-wiping response. Finally, pretreatment with i.ves. ruthenium red, a cation channel blocker, antagonized the excitatory and desensitizing effects of i.ves. RTX. This study demonstrates that repeated application of both CAP and RTX into the bladder produces behavioral effects indicative of local sensory afferent desensitization. I.ves. CAP and RTX appear to produce their excitatory and desensitizing effects via a common mechanism, which is dependent on cation channel activation. Furthermore, desensitization via i.ves. RTX administration appears to be specific to bladder afferents, occurs at low doses, and can be achieved over a broad dose range with minimal disruption of other behaviors. This study demonstrates for the first time that desensitization of sensory afferents in a deep structure can be produced via local application of RTX in awake, freely moving animals. Insofar as CAP-sensitive innervation regulates both pain and micturition in the human bladder, the i.ves. model in rats may be useful in assessing the utility of local afferent desensitization for the treatment of neuropathic bladder disorders.