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Trypanosoma cruzi infection, transmitted by insect vectors or blood transfusions, is an important cause of morbidity and mortality in many Latin American countries. Treatments are toxic and frequently ineffective in curing patients with chronic manifestations of the infection (Chagas disease). Potentially exploitable chemotherapeutic targets of T. cruzi are enzymes of the sterol biosynthesis pathway. In particular, the P450 enzyme, lanosterol 14alpha-demethylase, has been implicated as the target of azole antifungal drugs that have potent anti-T. cruzi activity. In the work reported here, the T. cruzi lanosterol 14alpha-demethylase (Tc14DM) gene was cloned by degenerate PCR. The gene was found to be expressed in both insect and mammalian life-cycle stages of the parasite. Tc14DM was able to complement the function of the homologous gene in yeast (erg11) as demonstrated by restored ergosterol production in an erg11-deficient yeast strain. When the yeast strain was co-transfected with the P450 reductase gene from Trypanosoma brucei, the amount of ergosterol production was increased, indicating that the endogenous yeast P450 reductase was an inefficient partner with Tc14DM. Heterologous expression of Tc14DM in the baculovirus/Sf9 system resulted in a 52kDa product. The protein was observed to have the characteristic absorbance spectra of a P450 enzyme. A typical Type II binding spectrum was produced when the imidazole compound, ketoconazole, was mixed with the Tc14DM, demonstrating that ketoconazole binds the enzyme.