In order to determine the actual distance between the active site and the substrate binding site, termed the basic protrusion, of Escherichia coli ribonuclease HI, synthetic oligonucleotide duplexes with gradually extended overhangs were used, in which the enzymatic cleavage was restricted to a single site with 2'-O-methylnucleosides. The affinity of the enzyme for each substrate was determined by kinetic analysis. It was found that the affinity increased markedly when one nucleotide was attached to the 3' end of the DNA strand of the nine-base-pair hybrid duplex and then increased slightly as the DNA strand was extended further, whereas elongation of the strand in the other direction caused no change. When a mutant enzyme, in which three lysine residues in the basic protrusion were altered to alanine, was used, no increase in the kcat/K(m) value was observed. The results indicate that, for the productive binding, the axis from the 3' to the 5' end of the RNA strand of the substrate duplex must be oriented in agreement with the direction from the active site to the basic protrusion of the enzyme. The distance between the active site and the basic protrusion in the enzyme-substrate complex was shorter than that anticipated in modeling studies. A dynamic structure refinement, referred to as the normal mode analysis, was carried out in order to simulate the fluctuations of the basic protrusion.