In a previous study, we showed that the RNA-binding domain of human RNase H1 is responsible for the positional preference for cleavage exhibited by the enzyme (Wu, H., Lima, W. F., and Crooke, S. T. (2001) J. Biol. Chem. 276, 23547-23553). Here, we identify the substituents on the heteroduplex substrate and the amino acid residues within the RNA-binding domain of human RNase H1 involved in positioning of the enzyme. The human RNase H1 cleavage patterns observed for heteroduplexes with various 3'-DNA/5'-RNA and 5'-DNA/3'-RNA termini indicate that the 5'-most cleavage site on the oligoribonucleotide is positioned 7 bp from the first 3'-DNA/5'-RNA base pair. The presence or absence of phosphate or hydroxyl groups at either the 3'-DNA or 5'-RNA terminus had no effect on the human RNase H1 cleavage pattern. Substitution of the 3'-deoxynucleotide with a ribonucleotide, 2'-methoxyethyl nucleotide, or mismatched deoxyribonucleotide resulted in the ablation of the 5'-most cleavage site on the oligoribonucleotide. Mutants in which Trp43 and Lys59-Lys60 of the RNA-binding domain were substituted with alanine showed a loss of the positional preference for cleavage. Comparison of the kcat, Km, and Kd for the alanine-substituted mutants with those for human RNase H1 suggests that Lys59 and Lys60 are involved in binding to the heteroduplex and that Trp43 is responsible for properly positioning the enzyme on the substrate for catalysis. These data suggest that Trp43, Lys59, and Lys60 constitute an extended nucleic binding surface for the RNA-binding domain of human RNase H1, with the entire interaction taking place at the 3'-DNA/5'-RNA pole of the heteroduplex. These results offer further insights into the interaction between human RNase H1 and the heteroduplex substrate as well as approaches to enhance the design of effective antisense oligonucleotides.