We describe the selection of a previously unreported 21-base pair insertion following codon 69 of the HIV-1 reverse transcriptase (RT) from a patient undergoing multiple nucleoside analogue therapy. This insertion was a direct duplication of the preceding 21 bases of HIV-RT, and was selected in a background of NRTI-resistance mutations including substitutions at RT codons 41, 67, 184, 210, and 215. Longitudinal genotypic and phenotypic resistance tests performed before and after selection of the insertion suggested that the insertion conferred an additional decrease in susceptibility to some nucleoside analogues, most notably didanosine, stavudine, abacavir, and tenofovir. However, phenotypic analysis of an insertion-containing site-directed mutant constructed in an HIV-1 HXB2 background revealed no direct association between the 21-base pair insertion and decreased susceptibility to NRTIs, suggesting that the insert requires the context of the patients' virus in order to confer resistance. These observations may offer new insight into the relative contribution of HIV-RT codon 69 insertion mutations to antiretroviral resistance.