Background: Visceral leishmaniasis is a severe and potentially fatal disease caused by protozoa of the genus Leishmania, transmitted by phlebotomine sandflies. In Europe and the Mediterranean region, L. infantum is the commonest agent of visceral leishmaniasis, causing a wide spectrum of clinical manifestations, including asymptomatic carriage, cutaneous lesions and severe visceral disease. Visceral leishmaniasis is more frequent in immunocompromised individuals and data obtained in experimental models of infection have highlighted the importance of the host immune response, namely the efficient activation of host's macrophages, in determining infection outcome. Conversely, few studies have addressed a possible contribution of parasite variability to this outcome. Methods: In this study, we compared three isolates of L. infantum regarding their capacity to grow in the organs of mice, the way they activate the host's macrophages and other components of the immune response and also their capacity to cope with host's antimicrobial mechanisms, namely reactive oxygen and nitrogen species. Results: We found that the three parasite strains significantly differed regarding the degree to which they induced nitric oxide synthase (NOS2) and arginase expression in infected macrophages and the pattern of cytokine production they induced in the host, resulting in different degrees of inflammatory response in infected livers. Additionally, the three strains also significantly differed in their in vitro susceptibility to reactive oxygen and nitrogen species. This variability was reflected in the capacity of each strain to persist and proliferate in the organs of wild-type as well as NOS2- and phagocyte oxidase- deficient mice. Conclusions: The results obtained in this study show that parasite strain variability is an important determinant of disease outcome in L. infantum visceral leishmaniasis, with relevant implications for studies on host-pathogen interaction and also for leishmanicidal drug development.