Transgenic potato (Solanum tuberosum cv Désirée) plants overexpressing a soybean (Glycine max) type 1 sterol methyltransferase (GmSMT1) cDNA were generated and used to study sterol biosynthesis in relation to the production of toxic glycoalkaloids. Transgenic plants displayed an increased total sterol level in both leaves and tubers, mainly due to increased levels of the 24-ethyl sterols isofucosterol and sitosterol. The higher total sterol level was due to increases in both free and esterified sterols. However, the level of free cholesterol, a nonalkylated sterol, was decreased. Associated with this was a decreased glycoalkaloid level in leaves and tubers, down to 41% and 63% of wild-type levels, respectively. The results show that glycoalkaloid biosynthesis can be down-regulated in transgenic potato plants by reducing the content of free nonalkylated sterols, and they support the view of cholesterol as a precursor in glycoalkaloid biosynthesis.Glycoalkaloids are a family of steroidal toxic secondary metabolites present in plants of the Solanaceae family. In cultivated potato (Solanum tuberosum) the main glycoalkaloids, ␣-chaconine and ␣-solanine, are triglycosylated products of the same aglycone, solanidine, but they differ in their sugar moieties (Friedman and McDonald, 1997). The highest glycoalkaloid level in potato plants is found in flowers and sprouts, followed by the leaves, and the lowest amounts are detected in stems and tubers. The amount of glycoalkaloids increases upon wounding and light exposure, something that may render tubers unsuitable for human consumption. Mild clinical symptoms of glycoalkaloid poisoning include abdominal pain, vomiting, and diarrhea, and an upper safe limit in tubers of 200 mg total glycoalkaloids (TGA) kg Ϫ1 fresh weight has been recommended by leading authorities. However, this upper limit is close to levels found in tubers destined for human consumption, and efforts should be made to keep TGA levels low when introducing new varieties on the market (see Valkonen et al., 1996).The biosynthesis of glycoalkaloids in potato is currently not fully understood. Solanidine has been proposed to be synthesized from the key precursor in plant sterol synthesis, cycloartenol, in a biosynthetic route including cholesterol, a sterol lacking alkylations at the C-24 position in the side chain (Heftmann, 1983; Bergenstråhle et al., 1996;Friedman and McDonald, 1997; Fig. 1). Cholesterol is in most plant species only a minor sterol, but is present at relatively high levels, approximately 15% to 20% of total sterols, in Solanaceous plants such as potato and tobacco (Nicotiana tabacum). One of the final reactions in the synthesis of glycoalkaloids is the glucosylation or galactosylation of solanidine to yield ␥-chaconine or ␥-solanine, respectively. A cDNA encoding the solanidine glucosyltransferase (SGT) enzyme has been cloned (Moehs et al., 1997). The SGT mRNA increased after wounding, in line with previous measurements of wound-induced SGT activity and glycoalkaloid levels. However, the ga...