Despite its clinical importance, the mechanisms that mediate or generate itch are poorly defined. The identification of pruritic compounds offers insight into understanding the molecular and cellular basis of itch. Imiquimod (IQ) is an agonist of Toll-like receptor 7 (TLR7) used to treat various infectious skin diseases such as genital warts, keratosis, and basal cell carcinoma. Itch is reportedly one of the major side effects developed during IQ treatments. We found that IQ acts as a potent itch-evoking compound (pruritogen) in mice via direct excitation of sensory neurons. Combined studies of scratching behavior, patch-clamp recording, and Ca(2+) response revealed the existence of a unique intracellular mechanism, which is independent of TLR7 as well as different from the mechanisms exploited by other well-characterized pruritogens. Nevertheless, as for other pruritogens, IQ requires the presence of transient receptor potential vanilloid 1 (TRPV1)-expressing neurons for itch-associated responses. Our data provide evidence supporting the hypothesis that there is a specific subset of TRPV1-expressing neurons that is equipped with diverse intracellular mechanisms that respond to histamine, chloroquine, and IQ.