The genome of Trypanosoma cruzi contains tandem arrays of alternating genes encoding amastin and tuzin. Amastin is a surface glycoprotein abundantly expressed on the intracellular mammalian amastigote form of the protozoan parasite, and tuzin is a G-like protein. We demonstrated previously that the amastin-tuzin gene cluster is polycistronically transcribed to an equal extent in all parasite life cycle stages. The steady state level of amastin mRNA, however, is 68-fold more abundant in amastigotes than in epimastigotes. Here we show that the half-life of amastin mRNA is 7 times longer in amastigotes than in epimastigotes. Linker replacement experiments demonstrate that the middle onethird of the 630-nucleotide 3-untranslated region (UTR) is responsible for the amastin mRNA up-regulation. This positive effect is dependent on the distance of the 3-UTR segment from the stop codon and the polyadenylation site as well as on its orientation. A protein or protein complex more abundant in amastigotes than in epimastigotes binds to this minimally defined 3-UTR segment and may be involved in its regulatory function.Trypanosoma cruzi is the protozoan parasite that causes Chagas' disease, a debilitating illness that is a major cause of morbidity and mortality in many parts of Latin America. During its life cycle, T. cruzi passes through three developmental stages. In their insect vectors, the parasites multiply as extracellular epimastigotes in the midgut, migrate to the hindgut, and then differentiate into nondividing trypomastigotes. These infective organisms are excreted in the feces after a blood meal. When they contaminate the puncture site or mucous membranes of a mammalian host, they can invade a variety of cell types. Once inside host cells, the trypomastigotes differentiate into amastigotes, which multiply in the cytoplasm. After many cycles of proliferation, the amastigotes differentiate into trypomastigotes and enter the circulation when the host cell ruptures. These trypomastigotes perpetuate either the infection or the life cycle when they invade other host cells or are ingested by insect vectors, respectively.Since the intracellular amastigotes are at the core of the persistence of T. cruzi infection, we previously sought to identify genes that are preferentially expressed by amastigotes in order to develop a better understanding of the molecular biology of this parasite form. We identified two novel genes, encoding the proteins amastin and tuzin, which occur in alternating tandemly arranged pairs (1, 2). Amastin is an abundant glycoprotein on the surface of amastigotes. Less is understood about tuzin's function or location, but it possesses sequence similarity to a heterotrimeric G-protein. Most tandem genes in T. cruzi and other trypanosomatids, such as Leishmania species and African trypanosomes, do not have recognizable upstream promoters and are transcribed into polycistronic precursor RNAs (3-6). The 5Ј ends of the resulting mature monocistronic mRNAs are defined by the trans-splicing of a 39-nucleotide ...