Flap endonuclease-1 or FEN-1 is a structure-specific and multifunctional nuclease critical for DNA replication, repair, and recombination; however, its interaction with DNA substrates has not been fully understood. In the current study, we have defined the borders of the interaction between the FEN-1 protein and its DNA substrates and identified six clusters of conserved positively charged amino acid residues, which are in direct contact with DNA substrate. To map further the corresponding interactions between FEN-1 residues and DNA substrates, we performed biochemical assays employing a series of flap DNA substrates lacking some structural components and a series of binding-deficient point mutants of FEN-1. It was revealed that Arg(47), Arg(70), and Lys(326)-Arg(327) of FEN-1 interact with the upstream duplex of DNA substrates, whereas Lys(244)-Arg(245) interact with the downstream duplex. This result indicates the orientation of the FEN-1-DNA interaction. Moreover, Arg(70) and Arg(47) were determined to interact with the sites around the 2nd nucleotide (Arg(70)) or the 5th/6th nucleotide (Arg(47)) of the template strand in the upstream duplex portion counting from the nick point of the flap substrate. Together with previously published data and the crystallographic ainformation from the FEN-1.DNA complex that we published recently (Chapados, B. R., Hosfield, D. J., Han, S., Qiu, J., Yelent, B., Shen, B., Tainer, J. A. (2004) Cell 116, 39-50) we are able to propose a reasonable model for how the human FEN-1 protein interacts with its DNA substrates.