In cell suspension cultures of the meadow rue Thalictrum tuberosum, biosynthesis of the anti-microbial alkaloid berberine can be induced by addition of methyl jasmonate to the culture medium. The activities of the four methyltransferases involved in the formation of berberine from L-tyrosine are increased in response to elicitor addition. Partial clones generated by RT-PCR with methyltransferase-specific primers were used as hybridization probes to isolate four cDNAs encoding O-methyltransferases from a cDNA library prepared from poly(A)+ RNA isolated from methyl jasmonate-induced cell suspension cultures of T. tuberosum. RNA gel blot hybridization indicated that the transcripts for the methyltransferases accumulated in response to addition of methyl jasmonate to the cell culture medium. The cDNAs were functionally expressed in Spodoptera frugiperda Sf9 cells and were shown to have varying and broad substrate specificities. A difference of a single amino acid residue between two of the enzymes was sufficient to alter the substrate specificity. The four cDNAs were expressed either as four homodimers or as six heterodimers by co-infection with all possible combinations of the four recombinant baculoviruses. These 10 isoforms thus produced displayed distinct substrate specificities and in some cases co-infection with two different recombinant baculoviruses led to the O-methylation of new substrates. The substrates that were O-methylated varied in structural complexity from simple catechols to phenylpropanoids, tetrahydrobenzylisoquinoline, protoberberine and tetrahydrophenethylisoquinoline alkaloids, suggesting that some biosynthetic enzymes may be common to both phenylpropanoid and alkaloid anabolism.