The alcohol dehydrogenase gene (NtADH) was previously isolated from tobacco BY2 suspension cultured cell. Expression of this gene was dramatically increased only during the early stationary phase, and the 5'-untranslated region (5'-UTR) was hypothesized to be involved in the stimulatory effect at the post-transcriptional level. In this paper, we investigated whether the NtADH 5'-UTR possesses the ability to positively enhance gene expression at the translational level. For easily estimating translational efficiency, we used beta-glucuronidase (GUS) gene as a reporter and tobacco BY2 cell, Arabidopsis thaliana T87 cell, and rice Oryza sativa suspension cultured cells as host cells in a transient assay system. Compared with the control plasmid pBI221, insertion of the NtADH 5'-UTR enhanced GUS expression levels about 30-to 100-fold and 30- to 60-fold in transiently transformed BY2 and T87 cells, respectively. However, in transiently transformed O. sativa cells, expression was barely enhanced. In comparison with the 5'-UTR of tobacco mosaic virus (Omega sequence), a known translational enhancer, the NtADH 5'-UTR enhanced translation to a similar level. Meanwhile, the translational efficiency was affected by the sequence context around the AUG initiation codon at the translational initiation step. Moreover, this NtADH 5'-UTR also worked in stable tobacco transformants. Therefore, it is expected that this 5'-UTR will serve as a powerful tool for enhancing foreign gene expression.