Rice tryptamine 5-hydroxylase (T5H) is the second enzyme in melatonin biosynthesis, catalyzing tryptamine into serotonin. Transgenic rice plants, in which the expression of endogenous T5H was either overexpressed or repressed, were examined for alteration in melatonin biosynthesis. Unexpectedly, the overexpression genotypes showed reduced levels of melatonin, while the repression genotypes had elevated levels with an average increase of fourfold. With regard to melatonin intermediates, tryptamine and serotonin levels decreased, but tryptophan and N-acetylserotonin were unaltered in the overexpression genotypes compared with the wild type. In contrast, the repression genotypes had sevenfold higher tryptamine levels than the wild type. In addition, tryptophan and 5-hydroxytryptophan were present at higher levels in the repression genotypes than in both the wild-type and the overexpression genotypes. The enhanced melatonin synthesis in the repression genotypes was closely associated with a transcriptional increase in TDC1. When these rice plants were challenged by oxidative stressors such as herbicides, much higher melatonin synthesis was also observed in the repression genotypes than in either the wild-type or overexpression genotypes. These results suggest that the tryptamine increase through the suppression of T5H plays an important signaling role in triggering melatonin biosynthesis in rice, although the exact role of tryptamine remains to be uncovered.