In order to identify the basis for the relaxed enantio-selectivity of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) and to evaluate possible cross-resistance patterns between L-nucleoside-, D-nucleoside- and non-nucleoside RT inhibitors, to be utilised in anti-HIV-1 combination therapy, we applied an in vitro approach based on the utilisation of six recom-binant HIV-1 RT mutants containing single amino acid substitutions known to confer Nevirapine resistance in treated patients. The mutants were compared on different RNA/DNA and DNA/DNA substrates to the wild type (wt) enzyme for their sensitivity towards inhibition by the D- and L-enantiomers of 2'-deoxy- and 2',3'-dideoxynucleoside triphosphate analogs. The results showed that the 3'-hydroxyl group of the L-(beta)-2'-deoxyribose moiety caused an unfavourable steric hindrance with critic residues in the HIV-1 RT active site and this steric barrier was increased by the Y181I mutation. Elimination of the 3'-hydroxyl group removed this hindrance and significantly improved binding to the HIV-1 RT wt and to the mutants. These results demonstrate the critical role of both the tyrosine 181 of RT and the 3'-position of the sugar ring, in chiral discrimination between D- and L-nucleoside triphosphates. Moreover, they provide an important rationale for the combination of D- and L-(beta)-dideoxynucleoside analogs with non-nucleoside RT inhibitors in anti-HIV chemotherapy, since non-nucleosideinhibitors resistance mutations did not confer cross-resistance to dideoxynucleoside analogs.