The lanosterol synthases from Trypanosoma cruzi, Trypanosoma brucei and Pneumocystis carinii, the lanosterol 14-demethylases from Trypanosoma brucei and Trypanosoma cruzi, and the cytochrome P450 reductase from Trypanosoma brucei were cloned and characterized. The two trypanosome lanosterol synthases showed a novel difference in protein sequence identity from that of other lanosterol synthases, which could be exploited for the development of specific antitrypanosome inhibitors. Yeast strains for expressing lanosterol 14alpha-demethylases were also developed. The first strains developed by tetrad dissection were time-consuming to produce, therefore another expression system was developed. The new system involved transforming the existing yeast strains, BJY1[pTb14DM] or BJY5[pTb14DM], and selection on FOA medium. With the addition of the T. brucei P450 reductase characterized in this study, the trypanosome lanosterol 14alpha-demethylases were able to regenerate their catalytic activity more efficiently than the strains containing only the native yeast P450 reductase. The various yeast strains developed in this study should be useful for screening antiparasite drugs. The BJY1[pTb14DM] (without reductase) and BJY5[pTb14DM] (with reductase) strains would also be useful in creating expression strains for other 14DM genes. Hopefully, these will be cloned in the near future.