The frequencies of multidrug resistance-associated mutations at codons 145, 151, and 69 of the human immunodeficiency virus (HIV) reverse transcriptase (RT) gene in strains from a group of 3,595 highly active antiretroviral therapy (HAART)-experienced patients were 0.22, 2.36, and 0.86%, respectively. Several amino acid substitutions different from the recently reported Gln145Met change (S. Paolucci, F. Baldanti, M. Tinelli, G. Maga, and G. Gerna, AIDS 17:924-927, 2003) were detected at position 145. Thus, amino acid substitutions selected at position 145 were introduced into the wild-type HIV type 1 (HIV-1) RT gene by site-directed mutagenesis, and recombinant HIV strains were assayed for their drug susceptibilities. Only Met and Leu substitutions at position 145 of the HIV-1 RT conferred multidrug resistance, while other amino acid changes did not. Lower levels of replication of the Gln145Met recombinant strain compared with those of both Gln151Met and wild-type recombinant strains were observed. In in vitro inhibition assays, expression and purification of the recombinant Gln145Met HIV-1 RT revealed a strong loss of catalytic efficiency of the mutated enzyme, as well as significant resistance to both zidovudine and efavirenz. Specific amino acid substitutions in the HIV RT nucleotide-binding pocket might affect both antiretroviral drug recognition and binding and decrease the level of virus replication, possibly by interfering with the enzyme activity. This finding may explain the lower frequency of Gln145Met/Leu mutations observed compared with the frequencies of Gln151Met/Leu mutations and the insertion at position 69 in HAART-experienced patients.