Different strains of Trypanosoma cruzi were transfected with an expression vector that allows the integration of green fluorescent protein (GFP) and red fluorescent protein (RFP) genes into the beta-tubulin locus by homologous recombination. The sites of integration of the GFP and RFP markers were determined by pulse-field gel electrophoresis and Southern blot analyses. Cloned cell lines selected from transfected epimastigote populations maintained high levels of fluorescent protein expression even after 6 months of in vitro culture of epimastigotes in the absence of drug selection. Fluorescent trypomastigotes and amastigotes were observed within Vero cells in culture as well as in hearts and diaphragms of infected mice. The infectivity of the GFP- and RFP-expressing parasites in tissue culture cells was comparable to wild type populations. Furthermore, GFP- and RFP-expressing parasites were able to produce similar levels of parasitemia in mice compared with wild type parasites. Cell cultures infected simultaneously with two cloned cell lines from the same parasite strain, each one expressing a distinct fluorescent marker, showed that at least two different parasites are able to infect the same cell. Double-infected cells were also detected when GFP- and RFP-expressing parasites were derived from strains belonging to two distinct T. cruzi lineages. These results show the usefulness of parasites expressing GFP and RFP for the study of various aspects of T. cruzi infection including the mechanisms of cell invasion, genetic exchange among parasites and the differential tissue distribution in animal models of Chagas disease.