The overall success of Trypanosoma cruzi depends on its ability to invade the host and establish a long-term infection. Little is known of the genetic factors responsible for observed differences in virulence from strain to strain in T. cruzi. A virulent T. cruzi line was derived from an attenuated parental line by two passages through mice. To identify virulence genes a subtraction library was constructed and screened for cDNA expressed exclusively in the virulent line. One cDNA hybridized to 3.5 and 4.5 Kb RNA present in virulent trypomastigotes but absent in attenuated trypomastigotes. Sequence analysis showed the cDNA to encode an 85 kDa protein with homology to members of the sialidase/trans-sialidase superfamily and has been designated vp85.1. The highest amino acid sequence similarity was to a previously described T. cruzi sialidase-homologue pseudogene [Takle, G.B., O'Conner, J., Young, A.J. and Cross, G.A.M. (1992) Mol. Biochem. Parasitol. 56, 117-128]. The vp85.1 amino acid sequence has higher homology to members of the 160 kDa flagellar-associated antigen family, FL-160, than to other 85 kDa expressed sialidase superfamily members. Southern blot analysis of virulent and attenuated lines demonstrated a complex hybridization pattern consistent with a multiple gene copy family that was identical in both lines. Antibody directed against recombinant vp85.1 peptide recognized proteins between 95 and 115 kDa in total virulent parasite lysates which were absent in attenuated lysates. Peptide N-glycosidase F treatment reduced the high molecular weight bands to 85 kDa, indicating vp85 is an N-linked glycoprotein. Immunofluorescence with anti-vp85.1 demonstrated surface localization of vp85.1 on virulent, but not attenuated, trypomastigotes. We postulate this new subfamily of trans-sialidases may play a role in virulence.