In order to identify a promoter system for high-level expression of transgenes in hairy roots, we characterized the chimeric super-promoter fused to the translational enhancer from tobacco etch virus (TEV). Transgenic tobacco plants and hairy roots were generated with the super-promoter:TEV sequence and a modified green fluorescence protein (mGFP5) as a reporter gene. To exploit the utility of hairy root cultures as a secretion-based expression system, the signal peptide of patatin was fused to mGFP5 to direct its secretion into the culture medium. Levels of mGFP5 RNA were on average sixfold higher in hairy roots than leaves. Likewise, GFP protein levels per gram of fresh weight were at least tenfold higher in hairy roots than leaves. Furthermore, more than 10% of the recombinant protein produced in the hairy root culture system was found in the medium. Immunoblotting with anti-GFP antibodies showed two products of 27.1 and 29.9 kDa in all leaf and hairy root tissue extracts, whereas a single 27.1-kDa product was detected in the medium. Inducibility of the promoter was studied with mature leaves and 14-day (midlog phase) hairy roots. A twofold increase in mRNA levels was found immediately after wounding in both mature leaves and hairy roots, with a corresponding increase in mGFP5 protein after 24 h. Our studies demonstrate the utility of the super-promoter:TEV system for high-level expression of recombinant proteins in hairy root bioreactors.