Plant peroxidases (EC 1.11.1.7) including horseradish peroxidase (HRP-Q, but not the nonplant peroxidases, are known to be highly specific indole-3-acetic acid (IAA) oxygenases which oxidize IAA in the absence Of H2O2, and superoxide anion radicals (O-2(.-)) are produced as by-products. Hypaphorine, a putative auxin antagonist isolated from ectomycorrhizal fungi, inhibited the IAA-dependent generation of O-2(.-) by HRP-C, which occurs in the absence of H2O2. Hypaphorine has no effect on the nonspecific heme-catalyzed O-2(.-) generation induced by high concentration of ethanol. It is probable that the inhibitory effect of hypaphorine on O-2(.-) generation is highly specific to the IAA-dependent reaction. The mode of inhibition of the IAA-dependent O(2)(.-)generating reaction by hypaphorine was analyzed with a double-reciprocal plot and determined to be competitive inhibition, indicating that hypaphorine competes with IAA by binding to the putative IAA binding site on HRP-C. This implies the importance of structural similarity between hypaphorine and IAA. This work presented the first evidence for antagonism between IAA and a structurally related fungal alkaloid on binding to a purified protein which shares some structural similarity with auxin-binding proteins.