Chalcone isomerase (CHI; EC 5.5.1.6) is a key enzyme in the flavonoid biosynthesis pathway. We isolated a CHI gene (SmCHI) from a cDNA library derived from Saussurea medusa (Asteraceae) cell cultures. The cDNA and genomic sequences of SmCHI are the same; in other words, this gene is intronless. The coding region of the gene is 699 bp long, and its deduced protein consists of 232 amino acids with a predicted molecular mass of 24 kDa and a pI of 4.7. The deduced amino acid sequence of SmCHI shares 79.3% identity with CHI from Callistephus chinensis, a familial relative to S. medusa; this homology is higher than those with CHI's from any other plant species. A functional bioassay for SmCHI was performed by transforming Nicotiana tabacum plants in the sense or antisense orientation under the regulation of the cauliflower mosaic virus (CaMV) 35S promoter. Transgenic tobacco plants overexpressing sense SmCHI produced up to fivefold total flavonoids over wild-type tobacco plants, mainly due to an enhanced accumulation of rutin. Transgenic tobacco plants with antisense SmCHI accumulated smaller amounts of flavonoids; this is apparently brought about by suppressed expression of the endogenous CHI gene. CHI activities also positively correlated with the amounts of total flavonoids accumulated in the transgenic plants. It is concluded that overexpression of SmCHI can be used as a useful approach to increase flavonoid production in transgenic plants.