Dietary intake of phytosterols (plant sterols) has been shown to be effective in reducing blood cholesterol levels, thereby reducing the risk of cardiovascular disease. Phytosterols are most commonly sourced from vegetable oils, where they are present as minor components. We report here the generation of transgenic tobacco seeds substantially enhanced in phytosterol content by the expression of a modified form of one of the key sterol biosynthetic enzymes, 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR). The constitutive expression of an N-terminal truncated Hevea brasiliensis HMGR (t-HMGR), lacking the membrane binding domain, enhanced seed HMGR activities by 11-fold, leading to increases in total seed sterol of 2.4-fold. Seed-specific expression of t-HMGR enhanced total seed sterol levels by 3.2-fold, to 1.36% dry weight or 3.25% of oil. 4-desmethylsterols were increased by 2.2-fold, whilst certain sterol biosynthetic intermediates, in particular cycloartenol and 24-ethylidene lophenol, also accumulated. The additional sterol in seed tissue was present in the form of fatty acid esters. Constitutive expression of t-HMGR increased leaf phytosterol sterol levels by 10-fold, representing 1.8% dry weight, and the sterol was sequestered, in acyl ester form, as cytoplasmic 'oil droplets'. These studies establish HMGR as a key enzyme controlling overall flux into the sterol biosynthesis pathway in seed tissue, but the accumulation of certain intermediates suggests additional slow steps in the pathway. The expression of an N-truncated HMGR activity has generated novel phytosterol-enriched raw materials that may provide the basis of new sourcing opportunities for this important class of cholesterol-lowering actives.