Abstract: Rice (Oryza sativa) N-acetylserotonin methyltransferase (osASMT), the last enzyme in the synthesis of melatonin, was expressed in Escherichia coli and purified. We then characterized its enzyme kinetics, which is the first time this has been performed in plants. Purified glutathione S-transferase (GST)-fused recombinant osASMT (GST-osASMT) and GST-free osASMT showed specific enzyme activities of 6.6 and 12.6 pmol/min per mg protein, respectively. When evaluated by the Lineweaver-Burk equation, GST-free osASMT exhibited a Km of 864 μm. An in vitro enzyme assay of purified osASMT showed melatonin formation to be proportional to the enzyme and substrate concentrations, as well as time. Unlike animal ASMT, high substrate concentrations did not inhibit the activity of osASMT. Finally, melatonin biosynthesis in rice seedlings was affected by light intensity, with etiolated shoots grown in continuous darkness producing more melatonin than shoots grown in continuous light. The level of melatonin in relation to the light intensity closely paralleled the mRNA level of osASMT in the shoots, suggesting that endogenous melatonin is upregulated in darkness, as is the case in animals.