Type I DNA topoisomerases homologous to Escherichia coli topoisomerase I normally only remove negative supercoils from DNA. Topoisomerases I from various eukaryotes share sequence homology and remove both positive and negative supercoils from DNA. Here we report that vaccinia virus topoisomerase I has significant difference in substrate preference from the other homologous type I topoisomerases. Vaccinia virus topoisomerase I shows a definite preference for removal of positive supercoils. In contrast, topoisomerase I from human, wheat germ and Saccharomyces cerevisiae has little preference between positive and negative supercoils. The vaccinia enzyme may have evolved for functions required for optimal viral growth. topoisomerases.