Thymidine analogue resistance mutations (TAMs) in HIV-1 reverse transcriptase (RT) associate in two clusters: (i) TAM1 (M41L, L210W and T215Y) and TAM2 (D67N, K70R, K219E/Q, and sometimes T215F). The amino acid substitution H208Y shows increased prevalence in patients treated with nucleoside analogues and is frequently associated with TAM1 mutations. We studied the molecular mechanism favoring the selection of H208Y in the presence of zidovudine, tenofovir and other nucleoside RT inhibitors (NRTIs). NRTI susceptibility was not affected by the addition of H208Y in phenotypic assays carried out in MT-4 cells using recombinant HIV-1 containing wild-type (subtype B, BH10), H208Y, M41L/L210W/T215Y or M41L/H208Y/L210W/T215Y RTs. However, enzymatic studies carried out with purified RTs revealed that in the presence of M41L/L210W/T215Y, H208Y increases the RT’s ability to unblock and extend primers terminated with zidovudine, tenofovir and in a lesser extent, stavudine. These effects were observed with DNA/DNA complexes (but not with RNA/DNA) and resulted from the higher ATP-dependent excision activity of the M41L/H208Y/L210W/T215Y RT compared with the M41L/L210W/T215Y mutant. The increased rescue efficiency of the M41L/H208Y/L210W/T215Y RT was observed in the presence of ATP but not with GTP or ITP. Molecular dynamics studies predict an alteration of the stacking interactions between Tyr215 and the adenine ring of ATP due to long-distance effects caused by tighter packaging of Tyr208 and Trp212. These studies provide a mechanistic explanation for the association of TAM-1 and H208Y mutations in viral isolates from patients treated with NRTIs.