The Human immunodeficiency virus (HIV) reverse transcriptase (RT) is a bifunctional enzyme having DNA polymerase activity and the ribonuclease (RNase H) activity. Although inhibitors that target the polymerase site are clinically available, no effective RNase H inhibitor exists. To screen RNase H inhibitors, an isolated RT RNase H fragment, p15-EC, that has insertion of a alpha-helical substrate-binding loop derived from Escherichia coli RNase HI has been often used (Keck & Marqusee, 1995). It is important to structurally characterize interaction of the p15-EC with Mg2+ and/or inhibitors. In particular, the intact RT RNase H domain fragment has been expected to undergo significant conformational changes upon Mg2+ interaction and exhibits different dynamics upon slight mutations (Pari et al., 2003). Thus, we have used a series of 1H-15N Heteronuclear Single Quantum Coherence (HSQC) experiments in combination with three-dimensional solution NMR experiments to clarify whether the p15-EC shows similar structural features to that of the intact RT RNH domain. We also identified the binding site of one of inhibitors, and compared Mg2+ effects on the inhibitor interaction.