Plant alkaloids have attracted much interest owing to their physiological properties. Argemone mexicana is a traditional medicinal plant which contains protoberberine, protopine and benzophenanthridine type alkaloids some of them with medicinal properties. Despite advance in chemical structure elucidation, the biosynthesis of benzylisoquinoline alkaloids is still incompletely characterized, due to the long pathways and in some cases the low expression of enzymes. In the present work, the construction of an EST library from roots of Argemone mexicana, led to the isolation of an FAD dependent oxygenase involved in the biosynthesis of protoberberines. In particular, this enzyme might be involved in the biosynthetic pathway derived from coreximine since this alkaloid was the preferred substrate and was also identified to be present in the plant. Furthermore two cytochromes P450 from the biosynthesis of stylopine, including the missing methylenedioxy bridge forming enzyme (S)-cheilanthifoline synthase were functionally expressed in Spodoptera frugiperda (Sf-9) cells.