By determination of the nucleotide sequence adjacent to the right border of T-DNA of the mikimopine-type Ri plasmid (pRi1724) in Agrobacterium rhizogenes, a new open reading frame (ORF) encoding 318 amino acids was found. A transcript of 1.35 kb derived from this ORF was observed in hairy roots of Ajuga reptans by northern blotting analysis. Including its own promoter and terminator, this ORF was isolated from the pRi1724 T-DNA and introduced into tobacco plants by the Agrobacterium-binary vector system. Since mikimopine, an opine and a stereoisomer of cucumopine, was accumulated in all organs of the transgenic tobacco plants, the new ORF was deduced to be the mikimopine synthase gene. For comparison, the nucleotide sequence of cucumopine synthase encoded on pRi2659 was also determined. No homology was found between mikimopine synthase and cucumopine synthase at the nucleotide, but partial homology was found at the amino acid level. Mikimopine synthase and cucumopine synthase produced by a protein expression system using E. coli catalyzed the synthesis of mikimopine and cucumopine from L-histidine and alpha-ketoglutaric acid, requiring NADH as a cofactor. These synthesized opines were identified by paper electrophoresis, TLC and HPLC analyses. The synthesized mikimopine or cucumopine could be degraded by A. rhizogenes strains harboring Ri plasmids encoding the respective catabolic enzyme.