Trypanosoma cruzi, the causative agent of Chagas disease, is transmitted by blood feeding triatomine bugs. As T. cruzi cycles between the insect vector and vertebrate host, it goes through several distinct developmental stages. The molecular mechanism that regulates these differentiation steps is poorly understood. We have initiated a systematic dissection of metacyclogenesis, the differentiation step from replicating non-infective epimastigotes to non-replicating infective metacyclic trypomastigotes. Our aim is to functionally characterise novel stage-regulated genes, using transfection-based approaches. We demonstrate that one of these genes, MET3, encodes a protein that localises to the nucleus and associates with nucleolar antigens. During metacyclogenesis the structure of the nucleus is modified: heterochromatin spreads as the nucleus changes shape from round to elongated, and nucleolar antigens are dispersed in the metacyclic nucleus. Our data show that while dispersal of nucleolar antigens occurs in all cells of a stationary phase culture, MET3 protein is expressed only in the subset of cells that have differentiated to metacyclics or to intermediate forms. By expressing MET3-GFP fusion proteins in the parasite, we identified two sequence elements that can independently direct localisation. To address function, we generated MET3 null mutants. These knockout cells produced significantly fewer fully developed metacyclics than wild-type controls. However, this phenotype could not be complemented with an ectopic copy of MET3. The reduced rate of metacyclogenesis may therefore be unrelated to the loss of MET3. The knockout cells are able to complete the entire life-cycle in vitro. This demonstrates that MET3 is not essential for development of the infective stage. RNA transcripts of the second gene, (MET2) are induced during metacyclogenesis. Recombinant MET2 protein associates with the kinetoplast when expressed in epimastigotes. We analysed in detail its genomic locus near a putative centromere on chromosome 3, and generated deletion mutants for further investigating its role in differentiation.