To obtain basic information such as interaction between the water molecule and amino acids in the active site of HIV-1 Reverse Transcriptase (HIV-1 RT), ab initio molecular orbital calculations and the two-layer ONIOM method were performed. The energetic results from different methods show that the ONIOM2 (MP2/6-311G:HF/6-31G//HF/6-31G:HF/3-21G) can provide reliable results on the orientation of the water molecule in the HIV-1 RT active site. The interaction between the water molecule and Asp186 was found to be the most preferable. The obtained results from ONIOM2 calculations indicated that the active site model system included six amino acid residues (Asp186, Asp185, Met184, Tyr183, Leu187, and Tyr188) leading a preferable representation of the environment surrounding the water molecule in the more realistic model. The water molecule presented in the active site tends to form H-bonding with Asp186, Tyr183, and Tyr188 as indicated by the distances of O4-H2 = 1.91 A, O3-H7 = 2.36 A, and O3-H17 = 1.73 A, respectively. The stability of this complex system brings to the foundation of the estimated binding energy approximately -15.8 kcal/mol or -8.1 kcal/mol which is more stabilized relative to the smallest model complex. These observations revealed that the water molecule forms both a hydrogen bond donor and a hydrogen bond acceptor in the cavity and plays an important role in the specific conformation of the active site of HIV-1 RT. The H-bonding is a rather strong interaction; thus, the water might induce the conformation of the active site to fit the catalysis process and helpfully attract dNTP to elongate the viral DNA in the replication process of this enzyme.