Trypanosoma cruzi is the parasite that causes Chagas disease, a chronic illness that affects 16-18 million people. Recently, the sequencing of T. cruzi genome was concluded. This accomplishment stimulated post-genomic projects aiming at elucidating the differential protein expression through the parasite life cycle. Proteomics is the most suitable methodology for this since T. cruzi protein expression regulation occurs at post-transcriptional level. In order to study the basic proteins from T. cruzi proteome, conditions for two-dimensional gel electrophoresis (2-DE) of epimastigote, trypomastigote and amastigote life forms were developed. It was necessary to optimize the 2-DE experimental conditions since in the alkaline pH range the gels normally presents spot streaking, low resolution and poor reproducibility. The final protocol, developed for epimastigotas, consisted of addition of 10% isopropanol to the IPG gel strip rehydration buffer, sample loading using the "paper bridge" method, use of paper strip embedded in DTT solution near the cathode and isoelectric focusing using the Multiphor II apparatus (GE Healthcare). A total of 10 spots from the epimastigote gel were identified by peptide mass fingerprinting. The identified proteins were phosphoglycerate kinase, prostaglandin F2a synthase, methionine peptide sulfoxide reductase, methylthioadenosin phosphorylase, protein disulfide isomerase, AKB ligase and four hypothetical proteins. The optimized 2-DE conditions were applied to the construction of trypomastigotes and amastigotas two dimensional maps. The resulting maps permitted the visualization of differences in protein expression among the proteomes. Finally, the "Two-in-one" 2-DE methodology for wide range pHs was tested and gave promising results that may be used in future studies on T. cruzi comparative protein expression. Support: TDR/WHO, FAPDF and CNPq.