Metacyclogenesis in Trypanosoma cruzi involves the differentiation of replicating non-infective epimastigotes into non-replicating metacyclic trypomastigotes. This pre-adapts parasites for infection of the mammalian host and is characterised by several morphological changes and structural alterations to the nucleus, including nucleolar disaggregation. Experimental investigation of these developmental processes has been hampered by a lack of robust molecular markers. Here, we describe the precise temporal expression of the T. cruzi-specific protein Met-III, in the genome reference strain CL Brener. Expression is restricted to metacyclics in the insect stages of the life-cycle and is rapidly down-regulated following invasion of mammalian cells. Met-III localises to dispersed foci typical of the disassembled nucleolus in metacyclics and to the discrete single nucleolus of cells soon after macrophage invasion. To identify elements that target Met-III, we generated a series of tagged green fluorescent protein fusion proteins and examined their sub-nuclear location in transformed parasites. These experiments demonstrated that amino and carboxyl terminal fragments, characterised by clusters of basic residues, could independently mediate nucleolar sequestration. To investigate the function of Met-III, we used gene deletion. This showed that Met-III is not required for the development of metacyclic trypomastigotes and that null mutants can complete the life-cycle in vitro.