Poly(ADP-ribosyl)ation by poly(ADP-ribose) polymerases regulates the interaction of many DNA damage and repair factors with sites of DNA strand lesions. The interaction of these factors with poly(ADP-ribose) (PAR) is mediated by specific domains, including the recently identified PAR-binding zinc finger (PBZ) domain. However, the mechanism governing these interactions is unclear. To better understand the PBZ-PAR interaction, we performed a detailed examination of the representative PBZ-containing protein involved in the DNA damage response, aprataxin polynucleotide-kinase-like factor (APLF), which possesses two tandem PBZ domains. Here we present structural and biochemical studies that identify Y381/Y386 and Y423/Y428 residues in the conserved C(M/P)Y and CYR motifs within each APLF PBZ domain that are critical for the interaction with the adenine ring of ADP-ribose. Basic residues (R387 and R429 in the first and second PBZ domains, respectively) coordinate additional interactions with the phosphate backbone of ADP-ribose, suggesting that APLF binds to multiple ADP-ribose residues along PAR polymers. These C(M/P)Y and CYR motifs form a basic/hydrophobic pocket within a variant zinc finger structure and are required for APLF recruitment to sites of DNA damage in vivo.