Selenium (Se) plays an indispensable role in human nutrition and has been implicated to have important health benefits, including being a cancer preventative agent. While different forms of Se vary in their anticarcinogenic efficacy, Se-methylselenocysteine (SeMSC) has been demonstrated to be one of the most effective chemopreventative compounds. Broccoli (Brassica oleracea var. italica) is known for its ability to accumulate high levels of Se with the majority of the selenoamino acids in the form of Semethylselenocysteine. Therefore, it serves as a good model to study the regulation of SeMSC accumulation in plants. A cDNA encoding selenocysteine Se-methyltransferase, the key enzyme responsible for SeMSC formation, was cloned from broccoli using a homocysteine S-methyltransferase gene probe from Arabidopsis (Arabidopsis thaliana). This clone, designated as BoSMT, was functionally expressed in Escherichia coli, and its identity was confirmed by its substrate specificity in the methylation of selenocysteine. The BoSMT gene represents a single copy sequence in the broccoli genome. Examination of BoSMT gene expression and SeMSC accumulation in response to selenate, selenite, and sulfate treatments showed that the BoSMT transcript and SeMSC synthesis were significantly up-regulated in plants exposed to selenate but were low in plants supplied with selenite. Simultaneous treatment of selenate with selenite significantly reduced SeMSC production. In addition, high levels of sulfate suppressed selenate uptake, resulting in a dramatic reduction of BoSMT mRNA level and SeMSC accumulation. Our results reveal that SeMSC accumulation closely correlated with the BoSMT gene expression and the total Se status in tissues and provide important information for maximizing the SeMSC production in this beneficial vegetable plant. Selenium (Se) is an essential micronutrient for animals and humans, although it was once known only for its toxicity (Draize and Beath, 1935;Schwarz and Foltz, 1957). Se is a component of many enzymes and proteins in mammals (Kryukov et al., 2003). The nutritional function of Se is fulfilled by selenoenzymes/selenoproteins such as glutathione peroxidases involved in antioxidant protection (Rotruck et al., 1973), thioredoxin reductases that mediate redox regulation (Tamura and Stadtman, 1996), and iodothyronine 5#-deiodinase involved in hormonal regulation of metabolism (Larsen and Berry, 1995). Se constitutes the active sites of these selenoenzymes as the noncanonical amino acid, selenocysteine, and is crucial for their biological functions (Stadtman, 1996;Driscoll and Copeland, 2003).In addition to its nutritional essentiality, Se has been implicated to have important health benefits. These include roles in reducing the incidence of some debilitating disorders, such as in improving male fertility and immune function (McKenzie et al., 2001;Foresta et al., 2002); in reducing viral infection (Beck et al., 2003); and in slowing the aging process (SorianoGarcia, 2004). More recently, a large body of convincing e...