The presence of human immunodeficiency virus type 1 (HIV-1) bearing mutations resistant to nucleosidic inhibitors of the viral reverse transcriptase (RT) derived from HIV-seropositive asymptomatic and untreated volunteer blood donors was examined. The RT amplicons of 32 specimens were analyzed by using a reverse hybridization line probe assay technique that detects resistance against zidovudine (3'-azido-3'-deoxythymidine [AZT], didanosine (2',3'-dideoxyinosine [ddI], zalcitabine (2',3'-dideoxycytidine [ddC]), and lamivudine ((-)-beta-L-2',3'-dideoxy-3'-thiacytidine [3TC]) at amino acid positions 41, 69, 70, 74, 184, and 215 of the HIV RT. One sample (brp004, subtype B) showed an AZT resistance secondary mutation at position K70R. Fifteen specimens revealed one or more sites of nonreactivity to both wild-type- and mutant-specific probes (dual nonreactivity). Samples were also submitted to RT direct sequencing and phylogenetic analysis. Nine of 32 specimens belonged to non-B subtypes (C, D, F, and F/B or B/F mosaics). Three of these non-B isolates, named brp004, brp063, and brp069, revealed three other relevant AZT resistance mutations-a T215F mutation and two M41L mutations, respectively-hidden by the nonreactivity to line probe assay strips on the respective codon regions. The isolate brp004 also carried a D67N AZT resistance mutation revealed by direct sequencing. No nonnucleosidic RT inhibitor-resistant mutation was found. The analysis revealed a frequency of 2.26 x 10(-4) mutations per nucleotide for independent samples related to RT resistance. These findings emphasize the magnitude of naturally occurring reservoirs of drug-resistant virus among untreated HIV-1-positive individuals in Brazil.