DNA polymerase and RNase H (RH) activities of HIV reverse transcriptase (RT) have been recognized as potential targets for antiretroviral therapy for more than 15 years. The development of medicines targeting the DNA polymerase activity has been highly successful, with currently 12 drugs approved for the treatment of HIV infection and more candidates in preclinical and clinical development. In contrast, the discovery of potent and selective inhibitors of HIV RH has been slow, and inhibitors of this enzyme function have yet to reach the clinical development stage. Selective HIV RH inhibitors are likely to provide significant clinical benefit in combination therapies, considering the high prevalence of HIV strains resistant to currently available antiretroviral therapies. Recent progress in a number of key areas has provided new impetus to the discovery of HIV RH inhibitors. High throughput assay systems based on fluorescence detection have been developed, which facilitate screening of inhibitor candidates. Substantial progress has been made in expression, purification, crystallisation and solution studies of HIV RT and RH, in particular with regards to aspects of structural dynamics. Crystal structures of active site binding and allosteric HIV RH inhibitors bound to HIV RT and RH have been obtained. Finally, an improved understanding of similarities and differences in enzymatic mechanisms between related nuclease enzymes has provided new concepts for achieving inhibitor selectivity. Together, these developments provide promising new starting points for the rational design of selective HIV RH inhibitors.