The short interspersed repetitive element (SIRE) of the nuclear genome of Trypanosoma cruzi was first detected when comparing the sequences of loci that encode the TcP2beta genes. The present study was designed to assess its distribution and organization in the nuclear genome of the parasite. Southern blots of genomic DNA from different strains demonstrated that each one possesses a defined and characteristic pattern of SIRE distribution. The conservation of the SIRE sequence in T. cruzi strains allowed the development of a rapid inter-SIRE PCR reaction that yields strain-specific amplicon profiles. In the T. cruzi CL Brener clone, we found 1500 copies of the element distributed in all chromosomes. 16 genomic fragments containing SIRE (SZs) were isolated and characterized. In fragments SZ10, SZ12 and SZ31, SIRE was linked to TcRel, a novel repeated sequence that constitutes the 3' end of vp85 genes. SIRE was also linked to an unknown open reading frame in fragments SZ14 and SZ23 which might be related to the subtelomeric regions of T. cruzi chromosomes. Further sequencing of SZ fragments revealed that SIRE was also linked to protein coding genes that have not yet been described in kinetoplastids such as the one coding for PRP22 helicase and a thimet oligopeptidase. To allow the rapid-generation genetic markers associated with SIRE, we developed a SIRE-bubble PCR reaction that provided several such markers for the construction of the physical map of chromosome XVI. The results herein demonstrate that SIRE-associated sites (SAS) may be of great help in physical mapping and interpretation of T. cruzi genomic sequence data.