Flap endonuclease-1 (FEN-1) is a critical enzyme for DNA replication and repair. Intensive studies have been carried out on its structure-specific nuclease activities and biological functions in yeast cells. However, its specific interactions with DNA substrates as an initial step of catalysis are not defined. An understanding of the ability of FEN-1 to recognize and bind a flap DNA substrate is critical for the elucidation of its molecular mechanism and for the explanation of possible pathological consequences resulting from its failure to bind DNA. Using human FEN-1 in this study, we identified two positively-charged amino acid residues, R47 and R70 in human FEN-1, as candidates responsible for substrate binding. Mutation of the R70 significantly reduced flap endonuclease activity and eliminated exonuclease activity. Mutation or protonation of R47 shifted cleavage sites with flap substrate and significantly reduced the exonuclease activity. We revealed that these alterations are due to the defects in DNA-protein interactions. Even though the effect of the single R47 mutation on binding activities is not as severe as R70A, its double mutation with D181 had a synergistic effect. Furthermore, the possible interaction sites of these positively charged residues with DNA substrates were discussed based on FEN-1 cleavage patterns using different substrates. Finally, data were provided to indicate that the observed negative effects of high concentration of Mg2+ on enzymatic activity are probably due to the competition between the arginine residues and metal ions with DNA substrate, since mutants were found to be less tolerant.