OBJECTIVES: To characterize methyltransferases involved in the biosynthesis of benzylisoquinoline alkaloids in Stephania intermedia. RESULTS: Three N-methyltransferases, SiCNMT1, SiCNMT2, SiCNMT3, and O-methyltransferase SiSOMT were identified in Stephania intermedia. Then, four methyltransferase genes were cloned into the pGEX-6P-1 vector. The recombinant vectors were transformed into Escherichia coli BL21(DE3) for expression and were functionally tested. SiCNMT1, SiCNMT2, and SiCNMT3 could methylate (R)-coclaurine to produce (R)-N-methylcoclaurine. SiCNMT2 further methylated the product of (R)-N-methylcoclaurine to produce (R)-magnocurarine. Similarly, (R)-norcoclaurine was continuously catalyzed to yield (R)-N-methylnorcoclaurine and (R)-N, N-dimethylnorcoclaurine by SiCNMT2. Furthermore, SiSOMT was shown to catalyze the conversion of (S)-scoulerine to (S)-tetrahydropalmatine. CONCLUSIONS: The key methyltransferases, which were in the last step biosynthesis of (R)-magnocurarine, (R)-N, N-dimethylnorcoclaurine and (S)-tetrahydropalmatine were revealed and their activities were verified in vitro. Four novel methyltransferases will be promising candidates for methylation of benzylisoquinoline alkaloids.