DNA topoisomerase I (topo I) is involved in the regulation of DNA supercoiling, gene transcription, recombination, and DNA repair. The anticancer agent camptothecin specifically targets topo I. The mechanisms responsible for the regulation of topo I in cells, however, are not known. This study demonstrates that c-Abl-dependent phosphorylation up-regulates topo I activity. The c-Abl SH3 domain bound directly to the N-terminal region of topo I. The results demonstrate that c-Abl phosphorylated topo I at Tyr268 in core subdomain II. c-Abl-mediated phosphorylation of topo I Tyr268 in vitro and in cells conferred activation of the topo I isomerase function. Moreover, activation of c-Abl by treatment of cells with ionizing radiation was associated with c-Abl-dependent phosphorylation of topo I and induction of topo I activity. The functional significance of the c-Abl/topo I interaction is supported by the findings that (i) mutant topo I(Y268F) exhibited loss of c-Abl-induced topo I activity, and (ii) c-Abl-/- cells were deficient in the accumulation of protein-linked DNA breaks. In addition, loss of topo I phosphorylation in c-Abl-deficient cells conferred resistance to camptothecin-induced apoptosis. These findings collectively support a model in which c-Abl-mediated phosphorylation of topo I is functionally important to topo I activity and sensitivity to topo I poisons.