Amastigote surface proteins of Trypanosoma cruzi are likely targets of both humoral and cell-mediated immune responses, however, few such molecules have been well studied. In this study, we have used modified RACE (rapid amplification of cDNA ends) and SOE (gene splicing by overlap extension) polymerase chain reaction strategies to clone the gene for the previously described 83 kDa amastigote surface protein of T. cruzi. Of the several clones obtained, only one clone, clone 4, was found to encode the 20 amino acid sequence originally reported by Pan and McMahon-Pratt (J Immunol 1989;143:1001-1008). The identity of the cloned gene with the 83 kDa amastigote surface protein was further confirmed by the reactivity of polyclonal antisera against the purified 83 kDa protein with the gene product expressed in E. coli. Sequence analyses revealed that this amastigote surface protein (ASP-2) has two conserved aspartic acid box motifs and the highly conserved VTVxNVxLYNR motif characteristic of bacterial and viral sialidases and the type III module of fibronectin, respectively. ASP-2 thus joins ASP-1 as a member of the amastigote surface expressed family of sialidase-like molecules having strong homology with family 2 of the sialidase/trans-sialidase gene superfamily of T. cruzi.