DNA fragments were obtained by the polymerase chain reaction (PCR) using genomic DNA from T. rangeli as template and oligonucleotide primers encoding the active site amino acids of cysteine proteinase. After amplification by PCR, several DNA products were observed. These were purified and used as templates for a second round of PCR. This resulted in two DNA products of 475 and 498 bp. The 498 bp DNA (Tr-CP) contained both the sense and antisense primer sequences, and encoded a polypeptide having substantial homology with eukaryotic cysteine proteinases. The other product (Tr-DMR), which lacked the antisense primer sequence, encoded a polypeptide having homology with the DNA mismatch repair gene from Saccharomyces cerevisiae. The overall homology of the Tr-CP-encoded polypeptide to the cysteine proteinases from other trypanosome species was 69% identity (T. cruzi) and 73% identity (T. brucei), respectively. Northern blot analysis revealed that the Tr-CP gene was specifically expressed in T. rangeli as a 1.7 kb mRNA. A karyotype map of the chromosomes was also performed using pulsed-field gradient gel electrophoresis and Southern blot hybridization with these genes. T. rangeli has 14 chromosome bands ranging from 350 kb to 1.6 Mb, which were fewer in number and smaller in size compared with those from T. cruzi. The Tr-CP fragment and the Tr-DMR fragment hybridized with equal intensity on chromosome 1 (350 kb) and chromosome 2 (470 kb), respectively. These results suggest that non-pathogenic T. rangeli contains a conserved gene corresponding to the cysteine proteinase or a closely related enzyme, and that there is more than one copy of this gene, each found on different chromosomes.