The Saccharomyces cerevisiae Lanosterol 14 alpha-demethylase (14DM) gene was overexpressed in S. cerevisiae using promoter sequences of the highly expressed S. cerevisiae glyceraldehyde-3-phosphate dehydrogenase (TDH3) gene. To investigate factors affecting 14DM overproduction, the levels of 14DM-specific RNAs, apoprotein, and heme protein, respectively, were determined and the 14DM-specific RNA levels compared with the RNA levels originating from the endogenous TDH gene(s). The quantitative measurements revealed that the 14DM steady-state RNA levels reached were some three- to five-fold below the theoretically expected values. With a view towards further improving expression of the 14DM gene, the spacing between the TDH3 promoter and the AUG was adjusted precisely and to rule out possible toxic effects exerted by the 14DM protein, the TDH3 promoter was placed under galactose regulation by introducing an UASG segment. Furthermore, the effects of the gene copy number on 14DM overproduction were investigated. From the analysis of the improved expression constructs five conclusions could be reached: (1) expression from the native 14DM gene is comparable to the expression driven by the TDH3 promoter-14DM fusion construct on single copy plasmid vectors; (2) expression from the TDH3 promoter-14DM construct on single-copy vectors is nearly as efficient as expression from the corresponding endogenous TDH3 gene; (3) the gene copy number has an effect on the relative expression levels of the TDH3 promoter-14DM constructs; (4) the steady-state amounts of protein produced are very nearly proportional to gene dosage; and (5) protein toxicity does not have a major impact on 14DM production. The maximum yield of 14DM was in the order of 7% of the total yeast protein and the maximum production of functional 14DM heme protein appears to be limited by the availability of heme.