Tryptophan decarboxylase (TDC) is a cytosolic enzyme that catalyzes an early step of the terpenoid indole alkaloid biosynthetic pathway by decarboxylation of L-tryptophan to produce the protoalkaloid tryptamine. In the present study, recombinant TDC was targeted to the chloroplast, cytosol, and endoplasmic reticulum (ER) of tobacco (Nicotiana tabacum) plants to evaluate the effects of subcellular compartmentation on the accumulation of functional enzyme and its corresponding enzymatic product. TDC accumulation and in vivo function was significantly affected by the subcellular localization. Immunoblot analysis demonstrated that chloroplast-targeted TDC had improved accumulation and/or stability when compared with the cytosolic enzyme. Because ER-targeted TDC was not detectable by immunoblot analysis and tryptamine levels found in transient expression studies and in transgenic plants were low, it was concluded that the recombinant TDC was most likely unstable if ER retained. Targeting TDC to the chloroplast stroma resulted in the highest accumulation level of tryptamine so far reported in the literature for studies on heterologous TDC expression in tobacco. However, plants accumulating high levels of functional TDC in the chloroplast developed a lesion-mimic phenotype that was probably triggered by the relatively high accumulation of tryptamine in this compartment. We demonstrate that subcellular targeting may provide a useful strategy for enhancing accumulation and/or stability of enzymes involved in secondary metabolism and to divert metabolic flux toward desired end products. However, metabolic engineering of plants is a very demanding task because unexpected, and possibly unwanted, effects may be observed on plant metabolism and/or phenotype.