Recently, we have reported that lysine 6, arginine 32, lysine 62 and lysine 74 do not play a direct role in the dsRNA cleavage activity of HPR, however lysine 6, lysine 74 and lysine 62 appear to be involved in general catalysis, structural integrity and stability and DNA helix unwinding activity of HPR. A number of non-basic residues that include glutamine 28, glycine 38, proline 42, aspartic acid 83 and alanine 122 have been shown to be important for dsRNA cleavage activity of pancreatic RNases of primates. We and others have shown earlier that apart from the basic residues, one non-basic residue, glycine 38 is also important for the dsRNA cleavage activity of HPR. RNase A lacks glycine at position 38 and instead it has an arginine at that position. Mutating glycine 38 to alanine caused a decrease in the activity of HPR on dsRNA. It was proposed that the presence of glycine at position 38 improves the flexibility of the active site cleft which in turn improves the activity of HPR and BS-RNase on dsRNA. In douc langur pancreatic ribonuclease, which has similar dsRNA cleavage activity as that of HPR, glutamine 28 to leucine mutation has been shown to decrease the dsRNA cleavage activity by about 3-fold, similar to the glycine 38 to alanine mutation in HPR. On the other hand, this mutation in bovine background increases dsRNA cleavage activity by about 4-fold. In addition, arginine 39 which is located near the active site residue lysine 41 and present in RNase A as well appears to be important for the dsRNA cleavage activity of HPR. In this study we have investigated the importance of arginine 39 and glutamine 28 for the dsRNA cleavage activity of HPR. Based on the earlier studies, importance of glycine 38 has also been investigated in the context of arginine 39 and glutamine 28. Using nine variants of HPR namely Q28A, Q28L, G38D, R39A, Q28A/R39A, Q28L/R39A, R39A/G38D, Q28A/G38D and Q28A/G38D/R39A, this study demonstrates that arginine 39 is crucial for the dsRNA melting activity, and for this activity, at position 38 glycine is required. Both these residues are not directly involved in the RNA cleavage activity. At position 28, in place of glutamine, leucine is tolerated whereas alanine is detrimental for the dsRNA melting activity of HPR. HPR is a member of single strand preferring ribonuclease A superfamily. However, HPR MK-1775 exhibits a remarkably high activity against AMN107 double stranded RNA. This high dsRNA cleavage activity of HPR suggests the enzyme to be playing a role in host defense. In this study we have investigated the role of glutamine 28, glycine 38 and arginine 39 in the dsRNA cleavage activity of HPR. RNase A has a series of base and phosphate binding sites, in addition to the catalytic site, that help in its binding to RNA.