Genetic vaccination has been shown to be an effective means for controlling Trypanosoma cruzi infection and development of Chagas disease, yet to date only six genes have been tested including five members of the trans-sialidase (TS) gene family. Vaccination with these genes generates an immune response able to significantly control the infection, ho wever parasite clearance was not attained and in each study a small percentage of the animals did not survive. The goal of this study was to expand the diversity of genes assessed as genetic vaccine candidates from T. cruzi. Three non- TS genes were assessed as genetic vaccines, Tcb3, FCaBP, and LYT1. Although peptides from all three genes were found to be targets of cytotoxic T cell responses in chronically infected mice, cells that are critical for control of T. cruzi infection, only immunization with LYT1 protected mice from a normally lethal challenge of T. cruzi. As an alternative to testing individual T. cruzi genes as vaccines, pools of genes from the TS and mucin families were assessed in vaccination studies. The mucin family was selected because like the TS family it encodes GPI-anchored surface proteins that are abundantly expressed. We found that immunization with pools of TS but not mucin genes provided protection against a normally lethal challenge of T. cruzi.|Although Tcb3 was not effective as a genetic vaccine, its homology to the human b3 subunit of the AP-3 adaptor protein complex suggested it might play a role in protein trafficking in T. cruzi and prompted further investigation. No other adaptin molecules have been identified in T. cruzi and very little is known about protein trafficking in this organism. Unable to generate null mutants of Tcb3 in T. cruzi, we used its sequence to identify its homologue from T. brucei, Tbb3, and took advantage of the inducible RNAi expression system developed in T. brucei to express dsRNA for this gene. Parasites expressing dsRNA for Tbb3 were unable to complete cytokinesis and exhibited defects in flagellar adhesion.