Tyrosyl-DNA phosphodiesterase 1 (Tdp1) repairs topoisomerase I cleavage complexes (Top1cc) by hydrolyzing their 3'-phosphotyrosyl DNA bonds and repairs bleomycin-induced DNA damage by hydrolyzing 3'-phosphoglycolates. Yeast Tdp1 has also been implicated in the repair of topoisomerase II-DNA cleavage complexes (Top2cc). To determine whether vertebrate Tdp1 is involved in the repair of various DNA end-blocking lesions, we generated Tdp1 knock-out cells in chicken DT40 cells (Tdp1-/-) and Tdp1-complemented DT40 cells with human TDP1. We found that Tdp1-/- cells were not only hypersensitive to camptothecin and bleomycin but also to etoposide, methyl methanesulfonate (MMS), H(2)O(2), and ionizing radiation. We also show they were deficient in mitochondrial Tdp1 activity. In biochemical assays, recombinant human TDP1 was found to process 5'-phosphotyrosyl DNA ends when they mimic the 5'-overhangs of Top2cc. Tdp1 also processes 3'-deoxyribose phosphates generated from hydrolysis of abasic sites, which is consistent with the hypersensitivity of Tdp1-/- cells to MMS and H(2)O(2). Because recent studies established that CtIP together with BRCA1 also repairs topoisomerase-mediated DNA damage, we generated dual Tdp1-CtIP-deficient DT40 cells. Our results show that Tdp1 and CtIP act in parallel pathways for the repair of Top1cc and MMS-induced lesions but are epistatic for Top2cc. Together, our findings reveal a broad involvement of Tdp1 in DNA repair and clarify the role of human TDP1 in the repair of Top2-induced DNA damage.