Previous studies have shown that human topoisomerase I interacts directly with the tumor-suppressor protein p53. In the past few years it has repeatedly been suggested that topoisomerase I and p53 may play a joint role in the response to genotoxic stress. This led to the suggestion that p53 and human topoisomerase I may cooperate in the process of DNA repair and/or apoptosis. Recently we have demonstrated that a human topoisomerase I cleavage complex can be recognized by an additional topoisomerase I molecule and thereby form a so-called double cleavage complex. The double cleavage complex creates an about 13 nucleotides long single-stranded gap that may provide an entry site for recombinational repair events. Here we demonstrate that p53 stimulates both the DNA relaxation activity as well as the formation of the human topoisomerase I double cleavage complex by at least a factor of six. Stimulation of topoisomerase I activity by p53 is mediated via the central part of topoisomerase I. We also show that human, bovine, and murine p53 stimulate human topoisomerase I relaxation activity equally well. From these results it is conceivable that p53's stimulatory activity on topoisomerase I may play a role in DNA recombination and repair as well as in apoptosis.