The method most commonly used in screening of drugs for the treatment of Chagas' disease, microscopic counting of viable trypanosomes, is time-consuming, labor-intensive, and dependent on the observer. Although the tetrazolium dye [MTT; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay is comparatively quick and accurate, it requires careful attention in design as well as in interpretation of the results. Therefore, we examined under various conditions the sensitivity and specificity of the MTT assay versus microscopic counting for determination of the viability of Trypanosoma cruzi for drug-screening purposes. We tested different concentrations of MTT in phenazine methosulfate (PMS) against T. cruzi epimastigotes of the Y strain in different stages of logarithmic growth. In our model, in tests of benznidazole and nifurtimox the optimal concentration of MTT was 2.5 mg/ml of PMS and the optimal incubation period was 75 min. This method detected parasite concentrations of approx. 500,000 epimastigotes/ml (P<0.01), and the linear correlation between absorbance values and numbers of epimastigotes per milliliter was very strong (approx. R = 0.99). The present MTT assay results in faster determination of the activity of compounds, is more objective, and enables testing of several drugs simultaneously.