We report in this paper significant differences in the virulence of insect-derived and cultured metacyclic forms of Trypanosoma cruzi which are morphologically indistinguishable. Mice infected intraperitoneally with 103 metacyclic T. cruzi isolated from Rhodnius prolixus showed average parasitemia levels greater than 2 × 105 organisms/ml around day 10 post-infection (when first measured) and peak levels recorded on day 16 post-infection exceeded 4 × 107 organisms/ml. None of these animals survived after 30 days post-infection. In contrast, in mice infected with 103 or 104 metacyclic forms from axenic cultures the highest average parasitemia was approximately 104 organisms/ml and occurred around day 19 post-infection. In these animals, parasitemias declined with time to become undetectable and no mortality was recorded over the 100-day observation period. There was also a marked difference in the 50% lethal dose of insect- and culture-derived metacyclics. The value for the former was 670 parasites whereas none of the mice infected intraperitoneally with up to 106 cultured metacyclics died. These results point to a marked difference in the biological properties of insect-borne and cultured T. cruzi metacyclics under our experimental conditions and caution against extending results obtained with the latter to vector-transmissible metacyclics, at least in infectivity and virulence studies.