Nucleoside reverse transcriptase inhibitors (NRTI) are essential components of highly active antiretroviral treatment (HAART). Although several combinations can be used as NRTI backbones, not all are associated with good virological and/or immunological results. In particular, some NRTI combinations should be avoided due to antagonism (zidovudine plus stavudine) or to high rate of toxicity (didanosine plus stavudine). Tenofovir (TDF) and didanosine (ddI) are among the more often prescribed NRTI for their convenient posology (one pill each per day), relatively high genetic barrier for resistance, quite acceptable safety profile and remarkable antiviral potency when such drugs have been used as single drug or in combinations with other NRTIs. However, antiretroviral regimens containing TDF and ddI have been associated with a high rate of virological failure in HIV-infected naïve patients due to possible drug-interactions. The virological efficacy of this backbone in HIV-infected, HAART pre-treated subjects, is still controversial. Aim of this review is to assess the possible role that antiretroviral regimens containing TDF and ddI can have in the treatment of HIV-positive subjects, focusing on their plasmatic and/or intracellular interactions to optimize the antiretroviral efficacy and minimize the toxicities of this combination.