N-acetylserotonin methyltransferase (ASMT) is the final enzyme in a biosynthetic pathway that produces melatonin. ASMT may play a rate-limiting role in the production of this bioactive molecule in plants. There are three ASMT genes (ASMT1-ASMT3) in the rice genome, but only ASMT1 has been functionally characterized. A major barrier to further progress in characterizing these genes has been a failure of functional expression within the Escherichia coli. Purified recombinant ASMT2 and ASMT3 are inactive in ASMT enzyme catalysis. To determine the biological functions of ASMT2 and ASMT3, we first overexpressed them in rice calli, which resulted in enhanced production of melatonin in the respective transgenic calli. To further corroborate the functions of ASMT2 and ASMT3 as ASMT genes, we generated stable transgenic rice plants. ASMT enzyme activity was increased in comparison with the wild type in T2 homozygous transgenic rice plants expressing three ASMT genes independently. When seedlings were treated with 1 mm N-acetylserotonin (NAS), leaf melatonin contents were higher in the three transgenic lines than in the wild type. There were no significant differences between the transgenic lines and the wild type without this treatment. ASMT1 and ASMT2 transcripts were highly expressed in stems and flowers, but ASMT3 was barely detectable in any of the plant organs. All three ASMT mRNAs were simultaneously induced in treatments with abscisic and methyl jasmonic acids.