Recently, we reported that momordin I from Ampelopsis radix is an activator protein-1 (AP-1) function inhibitor. Mormordin I showed both inhibition of AP-1 transcriptional activity and cellular cytotoxicity. Moreover, its inhibitory action was also identified in the animal experiments. The present study attempted to determine the mechanism of the action of momordin I more clearly. In mouse fibroblast cells, momordin I suppressed the AP-1 activity induced by phorbol 12-myristate 13-acetate (PMA), as well as blocked the de novo synthesis of AP-1 protein. Momordin I also showed cell cytotoxic activities on various cell lines and blocked the uptake of [(3)H]thymidine in cell proliferation induced by PMA. From these results, the inhibitory action of momordin I on AP-1 was clearly identified. Through the competitive binding assays, it appears that the inhibitory site of momordin I might be in the Jun/Fos dimer, and not in the DNA. Momordin I also showed the inhibitory action on the Jun/Jun homodimer, as well as on the Jun/Fos heterodimer. It also inhibited the autoregulated de novo synthesis of AP-1 by the Jun/Jun homodimer. In addition, since the truncated AP-1 used in this study only has the bZip regions of c-Jun and c-Fos, it appears that the inhibitory action site of momordin I may be the basic region of c-Jun instead of on the same region of c-Fos.