Chagas disease remains an important health problem in Central and South America. Nitroimidazole derivative drugs like Benznidazole are commonly used to treat Trypanosoma cruzi infection. Natural variation of drug susceptibility between various T. cruzi stocks has been proposed as a possible explanation of treatment failure. Thus, the aim of this work was to determine potential correlations between in vitro Benznidazole susceptibility of different T. cruzi stocks and their genetic diversity. For this purpose, 16 natural stocks representing the overall genetic diversity of the parasite were analysed. Genetic characterisation was assessed by both random amplified polymorphic DNA (RAPD) and multilocus enzyme electrophoresis (MLEE) analyses. Drug activity was determined by two complementary methods, the MTT-PMS micro-method and FACs analysis. The 50% inhibitory concentrations (IC(50)s) were determined. Important variation of IC(50) values (7.3-16.9 microM) among stocks belonging to different discrete typing units (DTUs) was recorded. Further, correlation analysis showed that natural susceptibility to Benznidazole in T. cruzi expressed as IC(50) level was not related with its genetic structure represented by the different DTUs. These results are discussed in relation with the proposed hypothesis establishing a link between genetic diversity and biological behaviour in T. cruzi.