Objective Through heterologous expression of the tetrahydrocannabinolic acid synthase (THCAS) coding sequence from Cannabis sativa L. in Nicotiana benthamiana, we evaluated a transient plant-based expression system for the production of enzymes involved in cannabinoid biosynthesis. Results Thcas was modularized according to the GoldenBraid grammar and its expression tested upon alternative subcellular localization of the encoded catalyst with and without fusion to a fluorescent protein. THCAS was detected only when ER targeting was used; cytosolic and plastidal localization resulted in no detectable protein. Moreover, THCAS seems to be glycosylated in N. benthamiana, suggesting that this modification might have an influence on the stability of the protein. Activity assays with cannabigerolic acid as a substrate showed that the recombinant enzyme produced not only THCA (123 +/- 12 fkat g(FW)(-1) activity towards THCA production) but also cannabichromenic acid (CBCA; 31 +/- 2.6 fkat g(FW)(-1) activity towards CBCA production). Conclusion Nicotiana benthamiana is a suitable host for the generation of cannabinoid producing enzymes. To attain whole pathway integration, careful analysis of subcellular localization is necessary.