An extended spectrum of HIV-1 reverse-transcriptase (RT) mutations in HAART-treated patients has been recently described. To verify the possible association of previously unreported RT mutations with a decrease of phenotypic susceptibility to nucleoside (NRTIs) and non-nucleoside (NNRTIs) RT inhibitors, the RT sequence of 328 HIV-1-positive patients (102 naïve and 226 treated with HAART participating in either the PhenGen or Genpherex study) was analyzed. All treated patients were tested at the time of therapeutic failure with both phenotypic (Antivirogram, Virco) and genotypic analyses (VircoGen); the frequency of RT substitutions (positions 1-240) with respect to consensus B was compared to that of naïve patients using a Chi-square test. Amino acid changes at 13 positions not included in the IAS list of resistance-associated mutations were detected more frequently in treated than in naïve subjects. The mutations involving 10 of these positions were associated with a reduced susceptibility to antiretroviral drugs; K20R, T39A, K43EQN, E203KD, H208Y, and D218E were correlated with NRTI resistance while mutations K101EQP, H221Y, K223EQ, L228HR were associated to NNRTI resistance. A correlation was found between K20R and lamivudine resistance (P = 0.006) while T39A (P = 0.005), K43EQN (<0.001), E203KD (P = 0.010), and H208Y (P = < 0.001) seemed to be associated with a previous use of zidovudine and stavudine and with the development of thymidine analog resistance. For H208Y, an association with use/resistance to abacavir (P = 0.004) was also noted. D218E showed a weak association to didanosine resistance (P = 0.013). The data confirm that previously unreported mutations are associated with antiretroviral drug experience and, more importantly, with a reduced susceptibility to NRTIs and NNRTIs.