HIV reverse transcriptase (HIV-RT) contains two distinct protein domains catalyzing DNA polymerase and RNase H activities. Non-nucleoside reverse transcriptase inhibitor (NNRTI) binding to HIV-RT can affect RNase H activity. The structurally diverse NNRTIs capravirine, efavirenz, GW8248, TMC-125, and nevirapine all inhibited 5'-RNA directed HIV RNase H activity as partial inhibitors with maximal inhibition of 40-65%. Potencies of RNase H inhibition correlated with the respective potencies of DNA polymerase inhibition. Mutations in the NNRTI binding site (K103N, Y181C, Y188L, and K103N/Y181C) reduced the potency of RNase H inhibition, similar to their effects on DNA polymerase activity. The NNRTIs did not affect the activity of the isolated HIV RNase H domain. In contrast, 3'-DNA directed RNase H activity of HIV-RT was mechanistically distinct from 5'-RNA directed RNase H activity and was stimulated rather than inhibited by NNRTI binding to HIV-RT. Therefore, NNRTI binding to the polymerase domain of HIV-RT interferes with RNase H activity through a long-range effect, which is affected by the structure of the RNA:DNA hybrid substrate, but is independent of NNRTI compound structure and nucleic acid substrate sequence. (c) 2006 Elsevier Inc. All rights reserved.