DNA topoisomerases I are ubiquitous enzymes that play a crucial role in DNA condensation, replication, transcription, and repair. Eukaryotic enzymes are highly conserved and specifically targeted by natural anticancer agents such as camptothecin and its derivatives. These drugs poison top 1 by inhibiting the enzyme via trapping of top 1 clivage complexes, which ultimately generate cell death. New camptothecin derivatives with better pharmacologic characteristics are under development. Understanding top 1 functions and structure will help to discover more specific and less toxic top 1 inhibitors in order to circumvent drug resistance.