ABSTRACT The aim of the study was to analyze the relationship between genotypic and phenotypic drug resistance profiles of human immunodeficiency virus type 1 (HIV-1) strains isolated from patients during double-analogue nucleoside therapy. A drug-resistant HIV strain was isolated from 20 out of 25 patients, with 16 (64%) subjects carrying a virus with multiple drug resistance mutations. The most frequent resistance mutations were M184V (18 isolates) and M41L (7 isolates). Discordance between the genotypic and phenotypic profile for at least one drug was detected in 16 out of 25 strains. Particularly, eight isolates had a discordant genotypic-phenotypic resistance pattern for two drugs and one isolate had such a pattern for three drugs. A genotypic resistance pattern with a phenotypic sensitivity profile was detected in six isolates (four resistant to zidovudine and two resistant to lamivudine). On the other hand for several strains a genotypic pattern of sensitivity pattern to abacavir (10 strains), didanosine (7 strains), stavudine (3 strains), zidovudine (2 strains), and lamivudine (1 strain) with a phenotypic resistance profile was detected. After a follow-up period of 8 months, an impairment of virological and immunological parameters was detected only in subjects with an HIV-1 isolate with a phenotypic resistance profile in despite of the genotypic results. Predicting resistance phenotype from genotypic data has important limitations. Despite the low number of patients and the short follow-up period, this study suggests that during failing therapy with analogue nucleosides, a phenotypic analysis could be performed in spite of an HIV genotypic sensitivity pattern.