A novel protein from the parasite Trypanosoma cruzi homologous to calcineurin (serine-threonine phosphatase 2B) was identified and characterized. The Calcineurin A gene is present as a single copy gene per haploid genome and encodes a protein of 43 kDa that is expressed in all major developmental stages of T. cruzi. Surprisingly, it is mainly localized in the cell nucleus, in sharp contrast with its mammalian counterpart. The T. cruzi calcineurin A protein presents the three invariants motifs characteristic of the PPP serine-threonine phosphatase superfamily. However, out of the four domains typically present in all calcineurin described to date, the T. cruzi calcineurin A possess only two domains: the catalytic and the calcineurin B binding domain. Sequence similarity searches in the T. cruzi, Trypanosoma brucei and Leishmania major genomes revealed that only L. major presents a gene encoding a putative protein containing the four domains. On the other hand, the T. cruzi Calcineurin B subunit showed a conserved structure, and a reasonable level of similarity over the entire length with calcineurin B proteins from other organisms. Interaction between Calcineurin A and Calcineurin B was analyzed by yeast Two-Hybrid and GST pull-down assays.