The purpose of the present study was to investigate the involvement of chemical mediators, other than histamine, in the scratching behavior induced by H₃ antagonists. Scratching behavior was induced by the histamine H₃ antagonists iodophenpropit and clobenpropit (10 nmol/site) when they were injected intradermally into the rostral part of the back of mast-cell-deficient (WBB6F1 W/W^v) and wild-type (WBB6F1 +/+) mice. Subsequently, the effect of spantide, a tachykinin NK₁ antagonist, was measured for 60 min. The effects of the H₃ antagonists on in vitro histamine release from rat peritoneal mast cells were also investigated. When spantide was injected intradermally at a dose of 0.5 nmol/site, it significantly inhibited the response. Furthermore, iodophenpropit and clobenpropit (10⁻⁶ – 10⁻⁸ M) did not induce histamine release in isolated rat peritoneal mast cells. Our results indicate that substance P is involved in the skin responses elicited by the histamine H₃ antagonists. Moreover, the fact that these histamine H₃ antagonists did not induce significant increases in the histamine release from rat peritoneal mast cells suggests that the histamine H₃ receptor may not be present in the peripheral cells considered in this study.