The enzymes 3-hydroxy-3-methylglutaryl CoA reductase (HMGR) and C24-sterol methyltransferase type 1 (SMT1) have been proposed to be key steps regulating carbon flux through the sterol biosynthesis pathway. To further examine this hypothesis, we co-expressed the catalytic domain of Hevea brasiliensis HMGR (tHMGR) and Nicotiana tabacum SMT1 in tobacco, under control of both constitutive and seed-specific promoters, resulting in increased accumulation of total sterol in seed tissue by 2.5- and 2.1-fold, respectively. This enhancement is greater than when tHMGR and SMT1 were expressed singularly where, for example, seed-specific expression enhanced total sterols by 1.6-fold. Significantly, the relative level of 4-desmethyl sterols (end-product sterols) was higher in seed co-expressing tHMGR and SMT1 from seed-specific promoters (79% of total sterols) than when co-expressed from constitutive promoters (59% of total sterols) and similar to wild-type seed (80% of total sterols). These results demonstrate that HMGR and SMT1 work in concert to control carbon flux into end-product sterols and that the sterol composition can be controlled by the temporal activity of the promoters driving transgene expression. In addition, constitutive expression of the transgenes resulted in elevated accumulation of substrates for C4-demethylation reactions, which indicates that one or several enzymes catalysing such reactions limit carbon flow to end-product sterols, at least in a physiological situation when the carbon flow is upregulated.