DNA topoisomerases are involved in DNA metabolism. These enzymes are inhibited by antimicrobial and antitumoral agents and might be important targets in the chemotherapy of diseases caused by parasites. We have cloned and characterized the gene encoding topoisomerase II from the monoxenic trypanosomatid Blastocrithidia culicis (BcTOP2). The BcTOP2 gene has a 3693 nucleotide-long open reading frame that encodes a 138 kDa polypeptide. The B. culicis topoisomerase II (BctopoII) amino-acid sequence shares high similarity (>74%) with topoisomerases from other trypanosomatids, and shares a lower similarity (41%) with other eukaryotic topoisomerases II from yeast to humans. BcTOP2 is a single copy gene and encodes a 4.4 kb mRNA. Western blotting of B. culicis extracts using the antiserum raised against a C-terminal portion of BctopoII showed a 138 kDa polypeptide. Immunolocalization assays showed that the antiserum recognized the nuclear topoisomerase II.